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Regulation of Cell Cycle Progression by Calcium/
Calmodulin-Dependent Pathways
CHRISTINA R. KAHL AND ANTHONY R. MEANS
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Many hormones, growth factors, and cytokines regulate pro-
liferation of their target cells. Perhaps the most universal
signaling cascades required for proliferative responses are
those initiated by transient rises in intracellular calcium
(Ca2ⴙ). The major intracellular receptor for Ca2ⴙ is calmod-
ulin (CaM). CaM is a small protein that contains four EF-hand
Ca2ⴙ binding sites and is highly conserved among eukaryotes.
In all organisms in which the CaM gene has been deleted, it is
essential. Although Ca2ⴙ/CaM is required for proliferation in
both unicellular and multicellular eukaryotes, the essential
targets of Ca2ⴙ/CaM-dependent pathways required for cell
proliferation remain elusive. Potential Ca2ⴙ/CaM-dependent
targets include the serine/threonine phosphatase calcineurin
and the family of multifunctional Ca2ⴙ/CaM-dependent pro-
I. Introduction
II. Role of Calcium (Ca2⫹) in Cell Proliferation
A. Requirement of Ca2⫹ for cell proliferation
B. Ca2⫹ signals and the cell cycle
C. Calmodulin (CaM), an intracellular Ca2⫹ receptor
III. Regulation of Cell Proliferation by CaM
A. CaM expression and the cell cycle
B. Genetic analysis of CaM function
C. Requirement of CaM for cell growth in culture
D. In vivo studies of CaM function during cell growth and
proliferation
E. Targets of CaM and cell proliferation
IV. Role of Calcineurin in Cell Proliferation
A. Calcineurin structure and biochemistry
B. Genetic analysis of calcineurin function
C. Calcineurin function in mammalian systems
V. Role of Ca2⫹/CaM-Dependent Kinases (CaMKs) in Cell
Proliferation
A. Structure and biochemistry of CaMKs
B. Genetic analysis of CaMK function
C. CaMK function in mammalian systems
VI. Conclusions and Perspectives
I. Introduction
C
2⫹
ALCIUM (Ca ) IS a universal second messenger that
regulates a number of diverse cellular processes in-
cluding cell proliferation, development, motility, secretion,
Abbreviations: Ca2⫹, Calcium; CaM, calmodulin; CaMK, Ca2⫹/CaM-
dependent kinase; CAMKK, Ca2⫹/CaM-dependent protein kinase ki-
nase; cdk, cyclin-dependent kinase; CKI, cdk inhibitor; FKBP, FK506-
binding protein; hsp, heat shock protein; MEF, mouse embryonic
fibroblast; NFAT, nuclear factor of activated T cells; NLS, nuclear lo-
calization sequence; NRK, normal rat kidney.
719
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Endocrine Reviews 24(6):719 –736
Copyright © 2003 by The Endocrine Society
doi: 10.1210/er.2003-0008
tein kinases. Whereas these enzymes are essential in Aspergil-
lus nidulans, they are not required under normal growth con-
ditions in yeast. However, in mammalian cells, studies
demonstrate that both types of enzymes contribute to the
regulation of cell cycle progression. Unfortunately, the mech-
anism by which Ca2ⴙ/CaM and its downstream targets, par-
ticularly calcineurin and the Ca2ⴙ/CaM-dependent protein ki-
nases, regulate key cell cycle-regulatory proteins, remains
enigmatic. By understanding how Ca2ⴙ/CaM regulates cell cy-
cle progression in normal mammalian cells, we may gain in-
sight into how hormones control cell division and how cancer
cells subvert the need for Ca2ⴙ and its downstream targets to
proliferate. (Endocrine Reviews 24: 719 –736, 2003)
and learning and memory (1, 2). How can a single ion carry
out such a vast array of complex cellular processes? Hor-
mones, growth factors, cytokines, and neurotransmitters all
elicit increases in intracellular calcium, but differences in the
temporal and spatial nature of the intracellular Ca2⫹ tran-
sients enable a cell to tailor its response to a given hormone
(3, 4). Ca2⫹ can act directly on target proteins or its effects can
be mediated via intracellular Ca2⫹ binding proteins. The
complex nature of Ca2⫹ signals and the myriad of Ca2⫹
binding proteins in cells allow a single cell to use Ca2⫹ signals
for its own unique functions. For example, a pancreatic aci-
nar cell uses Ca2⫹ signals at its apex to control the release of
secretory granules, whereas a neuron uses the frequency of
Ca2⫹ signals to regulate learning and memory. However, all
cells must grow and divide, and Ca2⫹ is universally required
for cell proliferation. Although much is known about the
many diverse Ca2⫹-dependent pathways regulating muscle
contraction, secretion, and learning and memory, the nature
of Ca2⫹-dependent pathways regulating cell growth and dif-
ferentiation remains poorly characterized.
Tremendous progress has been made in the last few de-
cades in understanding key pathways that regulate cell
growth and division. The cell cycle consists of four primary
phases: G1, the first gap phase; S phase, in which DNA
synthesis occurs; G2, the second gap phase; and M phase, or
mitosis, in which the chromosomes and cytoplasmic com-
ponents are divided between two daughter cells (Fig. 1). The
transitions between these cell cycle phases are tightly regu-
lated, and checkpoints during the cell cycle allow the cell to
determine whether all is well before proceeding to the next
cell cycle phase (5). For example, if DNA damage occurs
during G2, the cell will pause and repair its DNA before entry
into mitosis (6, 7). Understanding the pathways that regulate
these cell cycle transitions has been facilitated by studies in
 720 Endocrine Reviews, December 2003, 24(6):719 –736 Kahl and Means • Calcium/Calmodulin and the Cell Cycle

pendent on the binding of its partner cyclin. Second, cdk

activity is regulated by phosphorylation, both positively as
with activation loop phosphorylation and negatively as with
tyrosine phosphorylation. Third, some cdks are regulated by
the binding of cyclin-dependent kinase inhibitors (CKIs),
which associate with the cdk or cyclin/cdk complex, pre-
venting activation. The two classes of CKIs are the p21/p27
family, whose members associate with both cdk2 and cdk4,
and the p15/p16 family, whose members associate only with
cdk4/cdk6. Fourth, many of the cyclin/cdk complexes are
regulated by their subcellular localization with nuclear lo-
calization, allowing them access to their targets.
Although all cdks are regulated by these general mecha-
nisms, we will specifically discuss the regulatory mecha-
nisms of cyclin D/cdk4 complexes after growth factor stim-
FIG. 1. Schematic diagram of the mammalian cell cycle transitions.
ulation (Fig. 2) (12, 14, 22). Cyclin D1 expression is strictly
Cell cycle transitions are regulated by a series of cdks and their cyclin
partners. Upon reentry from quiescence (G0), cyclin D accumulates dependent on the presence of growth factors and its accu-
and associates with cdk4. Cyclin D/cdk4 complexes preferentially
phosphorylate pRb in mid-late G1. Then, in a sequential manner, pRb
is phosphorylated by cyclin E/cdk2 complexes. As cells enter S phase,
cyclin A/cdk2 complexes become activated. The transition from G2 to
mitosis is primarily regulated by the activity of cyclin B/cdc2. Ca2⫹/
CaM is required at two points during the reentry from quiescence,
early after mitogenic stimulation and later near the G1/S boundary.
Additionally, Ca2⫹/CaM is implicated in the G2/M transition, M phase
progression, and exit from mitosis.

unicellular eukaryotes, such as yeast. Importantly, homolo-

gous pathways have been identified in multicellular eu-
karyotes that also serve to control cell cycle progression.
One group of proteins that acts as a fundamental regulator
of cell cycle transitions is the cyclin-dependent kinases
(cdks). The cdks are a family of serine/threonine protein
kinases that are dependent upon cyclin binding for activity
(8 –10). Both the cdks and cyclins as well as their functions in
regulating the transitions between cell cycle phases are
highly conserved among eukaryotes. cdk Proteins were ini-
tially identified in a yeast mutant screen to identify temper-
ature-sensitive mutants that displayed dramatic cell division
defects at the restrictive temperature. The major cdk impli-
cated in cell cycle control in Saccharomyces cerevisiae is cdc28p
(11). This single cdk associates with different cyclin partners
during each stage of the yeast cell cycle. Importantly, the
regulation of the cell cycle by cyclins and cdks is conserved
throughout eukaryotes.
Mammalian cells contain multiple cdks and cyclins, which
act at different phases of the cell cycle (Fig. 1) (8 –10). As cells
progress through G1, cyclin D/cdk4 complexes are first ac-
tivated and phosphorylate the tumor suppressor protein,
retinoblastoma (pRb) (12–14). Next, cyclin E/cdk2 com- FIG. 2. Schematic diagram of cdk4 activation. The activity of cdk4
plexes phosphorylate pRb in a sequential manner after cyclin complexes is regulated in four distinct manners: 1) cyclin D binding,
D/cdk4 phosphorylation (15). The hyperphosphorylation of 2) CKI binding, 3) nuclear localization, and 4) phosphorylation. Ini-
pRb enables the activation of the family of E2F transcription tially, cdk4 is present in one of two complexes, a chaperone complex
containing hsp90 and cdc37 or a dimeric, inactive complex with p15/
factors, which, in turn, regulate the expression of numerous
p16. Upon mitogenic stimulation, cyclin D mRNA and protein levels
genes required for S phase progression (16 –18). As cells enter increase dramatically, which enables the assembly of cyclin D and
S phase, cyclin A/cdk2 becomes activated and remains ac- cdk4 complexes. Although the p21/p27 family of CKIs inhibit cdk2
tivated into G2 phase (19, 20). In late G2, cyclin B/cdc2 is activity, they are essential for the proper assembly of cyclin D/cdk4
activated, allowing entry into mitosis (21). complexes. Because neither cyclin D nor cdk4 have a canonical NLS,
the nuclear import of these complexes is also dependent on p21/p27
The activity of cdks is tightly regulated throughout the cell proteins present in the complex. After nuclear accumulation of cyclin
cycle. Four major types of regulation are common to the cdk D/cdk4, CAK (cdk activating kinase) phosphorylates cdk4 on Thr172,
family (8 –10, 12). First, the activation of cdks is strictly de- resulting in full activation of cdk4 complexes.

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 Kahl and Means • Calcium/Calmodulin and the Cell Cycle
mulation after mitogenic stimulation is regulated at the lev-
els of transcription, translation, and protein stability. How-
ever, before cyclin D accumulation, cdk4 exists in two major
complexes. Because cdk4 is unstable in its monomeric form,
cdk4 associates with heat shock protein 90 (hsp90) and cdc37
in a large chaperone complex. cdk4 Also exists in an inactive,
dimeric complex with p15/p16 proteins. Cyclin D1 accumu-
lation ultimately leads to assembly with cdk4. Although the
family of p21/p27 proteins bind cdk2 complexes inhibiting
kinase activity, low concentrations of p21/p27 bound to cy-
clin D/cdk4 do not significantly inhibit kinase activity. In-
deed, p21/p27 proteins actually appear to promote the ac-
tivation of cyclin D/cdk4 by three mechanisms: complex
assembly, nuclear import, and cyclin D stabilization. In
mouse embryonic fibroblasts (MEFs) null for both p21 and
p27, the amount of assembled cyclin D/cdk4 complexes is at
least 10-fold lower than their wild-type counterparts. Neither
cyclin D nor cdk4 possess a canonical nuclear localization
signal (NLS), and a second role for p21/p27 proteins is to
provide an NLS to mediate nuclear entry of the complex.
When cyclin D is overexpressed in p21/p27 double-null
MEFs, it remains cytoplasmic. Finally, cyclin D is stabilized
via its association into a complex with cdk4 and p21/p27.
Importantly, ectopic expression of either p21 or p27 into the
double-null p21/p27 MEFs restores these defects in cyclin
D/cdk4 assembly and nuclear import. After nuclear accu-
mulation of cyclin D/cdk4 complexes, cdk4 is phosphory-
lated on Thr172 by the nuclear enzyme cdk-activating kinase
to promote full kinase activation. This multileveled regula-
tion of cdk4 allows exquisite control over the activation of
cyclin D/cdk4 in G1. Therefore, in addition to phosphory-
lating pRb during G1, cyclin D/cdk4 complexes also act to
sequester p21/p27 proteins in late G1, promoting the acti-
vation of cdk2 complexes.
Importantly, disruptions in the regulation of cyclin/cdk
complexes are found in a wide variety of endocrine tumors.
For example, cyclin D1 overexpression occurs in some breast
cancers, and the cyclin D1 gene, previously referred to as the
PRAD1 oncogene, was initially identified for its role in some
parathyroid adenomas (23). In those tumors, a chromosomal
rearrangement placed the cyclin D1 locus adjacent to the
PTH locus. This rearrangement leads to dramatic increases
in cyclin D1 expression.
Regulation of cell proliferation is common to a wide va-
riety of hormones, growth factors, and cytokines. For a com-
prehensive description of the hormonal regulation of cell
cycle-regulatory proteins, we refer readers to a recent review
by Pestell et al. (24). Both steroid hormones and peptide
hormones alter the expression and/or activity of proteins
that are components of the cyclin/cdk complexes. Common
to all the regulatory transitions of the cell cycle is the ubiq-
uitous second messenger, Ca2⫹, and the universally impor-
tant Ca2⫹ intracellular receptor, calmodulin (CaM). In the
past several years, progress has been made in understanding
how Ca2⫹/CaM regulates cell cycle transitions and affects
the activation state of cdk complexes. In this review, we will
discuss the requirements for Ca2⫹ and CaM during cell pro-
liferation. Then, we will focus on two classes of Ca2⫹/CaM-
dependent enzymes that have been implicated in cell cycle
regulation in eukaryotes.
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Endocrine Reviews, December 2003, 24(6):719 –736 721
II. Role of Calcium (Ca2ⴙ) in Cell Proliferation
A. Requirement of Ca2⫹ for cell proliferation
In all eukaryotic cells, Ca2⫹ is required in both the extra-
cellular environment and intracellular stores for cell growth
and division. In mammalian cells, lowering of extracellular
Ca2⫹ from 1.0 mm to 0.1 mm led to a gradual decrease in the
rate of proliferation (25). Extracellular Ca2⫹ is required at
multiple distinct points in the cell cycle in mammalian cells.
When proliferating mouse or human fibroblasts were placed
into media containing low Ca2⫹, they ceased cellular division
and accumulated in G1 (26 –28). In BALBc/3T3 fibroblasts,
this G1 arrest was reversible, and returning the extracellular
Ca2⫹ content to normal levels enabled cells to undergo DNA
synthesis within hours (28). Cells were most sensitive to the
depletion of extracellular Ca2⫹ at two points during the cell
cycle, in early G1 and near the G1/S boundary (29). When
human fibroblasts were stimulated by growth factors, de-
pletion of extracellular Ca2⫹ anytime during the first 8 h after
stimulation resulted in an inhibition of DNA synthesis (30).
At later times, depletion of extracellular Ca2⫹ had no effect
on the ability of the cells to enter S phase. Again, these arrests
due to low Ca2⫹ were fully reversible as cells continue to
proliferate after the addition of normal Ca2⫹ levels to the
media.
This requirement for extracellular Ca2⫹ in growth and
proliferation is modulated by the degree of cellular trans-
formation. Indeed, neoplastic or transformed cells continued
to proliferate in Ca2⫹-deficient media (31, 32). In contrast to
their normal counterparts, human fibroblasts transformed
with the simian virus 40 proliferated normally in very low
extracellular Ca2⫹ concentrations (29, 33). Examination of
primary cells, preneoplastic cells, and neoplastic cells re-
vealed a gradient for extracellular Ca2⫹ levels required for
proliferation (34). Primary C3H mouse skin cells have re-
duced rates of DNA synthesis when extracellular Ca2⫹ is
lowered to 0.05– 0.1 mm, whereas preneoplastic C3H/
10T1/2 and MCA-C3H/10T1/2 type I mouse fibroblasts re-
quired a reduction to 0.01 mm extracellular Ca2⫹ to inhibit
DNA synthesis. Finally, the neoplastic MCA-C3H/1-T1/2
type III fibroblasts continued to proliferate with very low
extracellular Ca2⫹ levels. Additionally, the proliferative re-
sponses of liver tumor cell lines in low Ca2⫹ reflected their
tumorigenic potential (35). Therefore, the strict requirement
for extracellular Ca2⫹ is lost during neoplastic transforma-
tion, but how this change in extracellular Ca2⫹ dependence
affects intracellular Ca2⫹-dependent pathways is unknown.
In addition to the requirement for extracellular Ca2⫹, in-
tracellular Ca2⫹ stores are also required for cellular prolif-
eration in mammalian cells. Depletion of intracellular
inositol 1,4,5-triphosphate-sensitive Ca2⫹ stores with phar-
macological agents, such as thapsigargin or 2,5-di-tert-butyl-
hydroquinone, resulted in a cessation of cell division (36).
These agents block the Ca2⫹ pumping ATPase present in the
endoplasmic reticulum and result in a depletion of Ca2⫹
stores in the endoplasmic reticulum. The consequences of
intracellular Ca2⫹ pool depletion included inhibition of DNA
synthesis, protein synthesis, and nuclear transport (36 –38).
Depletion of intracellular Ca2⫹ stores resulted in the accu-
 722 Endocrine Reviews, December 2003, 24(6):719 –736
mulation of cells in a quiescent state, and upon removal of
thapsigargin or 2,5-di-tert-butyl-hydroquinone, cells reen-
tered S phase with the same kinetics as cells released from
quiescence (36). Furthermore, depletion of intracellular Ca2⫹
stores at any point during G1 to S resulted in an accumulation
of cells in a G0-like state even when cells have partially
replicated DNA (F. Riberio-Neto and A. R. Means, unpub-
lished data). Therefore, normal cells require both extracel-
lular and intracellular Ca2⫹ for proliferation, with cells being
the most sensitive to Ca2⫹ depletion during G1.
B. Ca2⫹ signals and the cell cycle
In cells, cytoplasmic Ca2⫹ transients are generated by re-
lease of Ca2⫹ from intracellular pools or by entry of Ca2⫹
from the extracellular environment via Ca2⫹ channels in the
plasma membrane. Ca2⫹ transients come in a variety of cat-
egories including elemental “blips/quarks,” slightly larger
“puffs/sparks,” which are restricted to small areas, or
“waves,” which involve the whole cell (1– 4, 39). In addition
to spatial difference in Ca2⫹ transients, both the amplitude
and frequency of Ca2⫹ transients can be modulated. Even
though Ca2⫹ is a ubiquitous second messenger, the temporal
and spatial complexity of Ca2⫹ transients enables a cell to use
Ca2⫹ signaling for a wide variety of physiological responses.
Although many hormones and growth factors cause intra-
cellular Ca2⫹ transients, the nature of the Ca2⫹ signal allows
a cell to decode stimuli from a wide variety of hormones.
Although these Ca2⫹ signals act to regulate innumerable
cellular pathways, we will focus the discussion on the role of
Ca2⫹ transients during the cell cycle.
During the cell cycle, Ca2⫹ transients have been charac-
terized during G1 and mitosis (40, 41). In rat liver epithelial
cells (T51B), epidermal growth factor stimulation resulted in
a rise in intracellular Ca2⫹, and this rise required Ca2⫹ in the
extracellular environment (42). When mouse C127 cells,
which are a nontransformed cell line derived from a mam-
mary tumor, were synchronized in mitosis, multiple Ca2⫹
transients were observed as they entered early G1. Whereas
in mid-G1 there were no detectable transients, Ca2⫹ tran-
sients resumed near the G1/S boundary (Christenson, M., M.
Poenie, and A. R. Means, unpublished observations). There-
fore, Ca2⫹ transients within a cell correspond to the same cell
cycle points in which extracellular Ca2⫹ is required, namely
early G1 and at the G1/S boundary.
Ca2⫹ transients are also evident during several stages of
mitotic progression, particularly at the metaphase/anaphase
transition and during cytokinesis (43, 44). In sea urchin eggs,
Ca2⫹ transients occurred at pronuclear migration, nuclear
envelope breakdown, the metaphase to anaphase transition,
and during cleavage (45). These transients at the metaphase
to anaphase transition have also been demonstrated in mam-
malian Ptk1 cells (46). Regardless of the size and duration of
a Ca2⫹ transient, the Ca2⫹ signal is transduced into cellular
consequences by direct binding of Ca2⫹ to targets or by Ca2⫹
binding to intracellular receptors, such as CaM, which relay
the signal to Ca2⫹/CaM-dependent targets.
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Kahl and Means • Calcium/Calmodulin and the Cell Cycle
C. Calmodulin (CaM), an intracellular Ca2⫹ receptor
In mammalian cells, CaM is a 148-amino acid, highly con-
served Ca2⫹ binding protein that contains four EF-hand Ca2⫹
binding motifs (47). Based on nuclear magnetic resonance
and crystal structures of CaM in the apo and Ca2⫹-bound
state, we know that Ca2⫹-bound CaM has a dumbbell shape
with two EF-hand motifs on either end connected by a central
helix (47, 48). Ca2⫹ binding exposes hydrophobic patches,
promoting interaction with target enzymes. A number of
crystal structures of Ca2⫹/CaM bound to target peptides
demonstrate that CaM wraps around the target peptide, en-
gulfing it (48 –52). For targets of Ca2⫹/CaM, this binding has
enormous consequences. As in the cases of calcineurin and
Ca2⫹/CaM-dependent protein kinase II (CaMKII), one gen-
eral mechanism by which Ca2⫹/CaM-binding activates its
target enzymes is through the relief of autoinhibition.
CaM regulates numerous intracellular enzymes that in-
clude phosphodiesterases, adenylyl cyclases, ion channels,
protein kinases, and protein phosphatases (47). Ca2⫹/CaM-
dependent pathways are involved in the regulation of a wide
variety of cellular processes including secretion, cell motility,
ion homeostasis, gene transcription, neurotransmission, and
metabolism. Hormone and neurotransmitter stimulation of
cells leads to a variety of Ca2⫹/CaM-mediated responses
(53). Initially, hormone receptors are activated, leading to an
intracellular Ca2⫹ rise. Ca2⫹ regulates some targets directly
and other targets indirectly through Ca2⫹ binding proteins,
such as CaM. Persistent stimulation can cause changes in the
subcellular distribution of CaM, which leads to changes in
Ca2⫹/CaM responsiveness in a given area of the cell. Long-
term stimulation can also lead to changes in the total amount
of CaM making a cell more or less sensitive to Ca2⫹ signals
(53).
III. Regulation of Cell Proliferation by CaM
A. CaM expression and the cell cycle
Intracellular CaM levels are regulated as cells progress
through the cell cycle. In Chinese hamster ovary-K1 cells,
CaM levels fell within the first hour after release from plateau
phase and then doubled at the G1/S boundary due to in-
creased protein synthesis (54, 55). Similarly, in normal hu-
man fibroblasts, CaM levels decreased in the first few hours
after mitogenic stimulation and then increased 2- to 4-fold at
the G1/S boundary (56). However, as these cells approached
senescence, these changes in CaM levels during G1 were lost
and CaM levels remained relatively constant. In addition to
these cell culture experiments, changes in CaM levels were
also found in in vivo models of proliferative responses. Dur-
ing liver regeneration, there was a wave of increased CaM in
the early prereplicative period, which was also due to new
protein synthesis (57). However, another group found an
increase in CaM mRNA as well as protein levels after partial
hepatectomy (58). Similar to findings in mammalian cells, in
Aspergillus nidulans, CaM levels also increased 2-fold just
before DNA synthesis (59). Therefore, the rise in CaM levels
just before S phase may be universal, but why CaM must
increase at this point in the cell cycle or what the target of
CaM is at the G1/S boundary remains unknown.
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle
Indeed, manipulation of CaM expression affects the pro-
liferative capacity of cells. In mammalian cells, overexpres-
sion of CaM by 2- to 4-fold using a bovine-papilloma-virus-
based expression vector caused an acceleration of cell
proliferation (60). In A. nidulans, overexpression of CaM ac-
celerated cell cycle reentry from sporulation and shortened
cell cycle length (61). In mammalian cells, the acceleration of
cell proliferation was due to a shortening of G1. Examination
of six independent cell lines that overexpressed CaM dem-
onstrated a linear relationship between CaM concentration
and rate of G1 progression (60, 62). Because a relationship
between the amount of CaM and cell cycle timing exists in
both A. nidulans and murine C127 cells, it suggests that this
correlation was not merely secondary to a transformed phe-
notype. In contrast, reduction of CaM levels using antisense
CaM resulted in a transient inhibition of cell proliferation
(62). Deletion of one of two functional CaM genes, CaMII,
from chicken DT40 lymphoma B cells reduced CaM expres-
sion by 60%, and these cells exhibited slower growth (63).
Therefore, the amount of CaM in a cell regulates proliferative
rates, primarily due to effects on G1.
The association between CaM levels and proliferation is
reflected in the relationships between CaM levels and
cellular transformation. Transformation of Swiss 3T3 cells
with simian virus 40 or normal rat kidney (NRK) cells with
Rous sarcoma virus resulted in increased CaM levels (64).
Additionally, in normal chicken embryo fibroblasts, trans-
formation with Rous sarcoma virus resulted in increased
CaM levels due to increased protein synthesis (65). NRK
cells infected with a temperature-sensitive, transformation
defective mutant avian sarcoma virus (tsLA23) possessed
more CaM than uninfected controls (66). However, upon
shift to 40 C, the temperature at which they behaved more
like nontransformed cells, CaM levels dropped to levels
similar to nontransformed cells. Rat fibroblasts trans-
formed with a variety of oncogenes in combination with
protein kinase C have increased CaM levels even in the
presence of an overall reduction in CaM mRNA transcripts
(67). These studies raise the question: do increases in CaM
contribute to the transformed phenotype or do increases
in CaM merely reflect the growth advantages of trans-
formed cells? Furthermore, how relevant are studies of
Ca2⫹/CaM-dependent signaling cascades in transformed
cells to their function in normal cells?
Studies involving transformed cells and in A. nidulans
indicate a reciprocal regulation between CaM levels and
Ca2⫹. In transformed rat fibroblasts, a reduction in extra-
cellular Ca2⫹ resulted in an increase in CaM expression,
whereas an increase in extracellular Ca2⫹ resulted in a
decrease in CaM expression (68). In chicken DT40 lym-
phoma cells, deletion of CaMII caused an increase in rest-
ing Ca2⫹ levels (63). In A. nidulans, induction of CaM
expression using the alcA (alcohol dehydrogenase A) gene
promoter led to a 10-fold reduction in the level of extra-
cellular Ca2⫹ required for growth (61). Although CaM
and Ca2⫹ appear to be reciprocally regulated, how these
changes regulate downstream Ca2⫹/CaM pathways re-
mains unknown.
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Endocrine Reviews, December 2003, 24(6):719 –736 723
B. Genetic analysis of CaM function
Organisms that facilitate genetic analysis, such as S. cer-
evisiae, Schizosaccharomyces pombe, A. nidulans, and Drosophila
melanogaster, all contain a unique CaM gene, which is essen-
tial (59, 69 –71). The requirement for CaM has been charac-
terized in both budding and fission yeast. In yeast, CaM was
localized to sites of cell growth and the SPB (spindle pole
body) (72, 73). Based on this localization of CaM, it is not
surprising that studies involving the disruption of CaM func-
tion have revealed a role for CaM in nuclear division and the
maintenance of cell polarity in yeast (74).
In S. cerevisiae, CaM is required for mitotic progression.
When the expression of the CaM gene, CMD1, was regulated
using the GAL1 promoter, repression of CaM resulted in
nuclear division defects with cells having short mitotic spin-
dles and increased chromosomal loss (75). Additionally,
some cells also demonstrated bud growth inhibition. Using
a temperature-sensitive allele of CaM, Davis (76) synchro-
nized cells in G1 and followed their progression through the
cell cycle. These yeast cells were viable until mitosis and
demonstrated defects in both chromosomal segregation and
cytokinesis. In studying multiple temperature-sensitive CaM
mutants, Ohya and Botstein (77) found that they sorted into
four complementation groups, each of which exhibited a
defect in actin organization, CaM localization, nuclear divi-
sion, or bud formation. Similar to budding yeast, CaM is
required for mitotic progression in fission yeast. When cells
with a temperature-sensitive allele of CaM were synchro-
nized in S phase and released, they progressed through DNA
synthesis and then lost viability in mitosis, with defects in
chromosomal segregation (72).
In the filamentous fungi A. nidulans, CaM is also critical for
the progression through the G2/M transition. Repression of
CaM expression using the alcA promoter arrested the ma-
jority of cells in G2 (61). When cells were released from a
temperature-sensitive block in late G2, cells with low levels
of CaM failed to activate NIMXcdc2 and progress through
mitosis (78).
The results from yeast and A. nidulans demonstrate a crit-
ical role for CaM for entry into and progression through
mitosis. However, in mammalian cells, the requirement for
Ca2⫹ is clearly linked to G1 progression as well as mitosis.
Therefore, why do unicellular eukaryotes, such as yeast and
fungi, not demonstrate G1 defects when CaM function is
disrupted? One possibility is that mammalian cells have
evolved complex mechanisms to regulate G1 progression
that are dependent on Ca2⫹, and those pathways may not
exist in yeast and fungi. Another possibility is that it may be
more difficult to study defects in Ca2⫹/CaM-dependent
pathways during G1 in unicellular eukaryotes.
C. Requirement of CaM for cell growth in culture
The importance of CaM for mammalian cell survival is
reflected by both the number of CaM genes in higher eu-
karyotes and its degree of evolutionary conservation (79).
Rodents and humans have three CaM genes on three sepa-
rate chromosomes (80 – 82). Strikingly, the encoded amino
acid sequences are not merely conserved among these sep-
 724 Endocrine Reviews, December 2003, 24(6):719 –736 Kahl and Means • Calcium/Calmodulin and the Cell Cycle

arate genes and species, they are identical. Because mammals Indeed, more than 80% of human cancers have a defect in the
have three CaM genes that encode identical proteins and cyclin D/cdk4/pRb/E2F pathway (22). Importantly, a can-
CaM is essential in genetic model systems, deletion of CaM cer that has an alteration in one component of this pathway
in mice would be not only labor intensive but also could be does not have defects in the other components. Therefore, in
uninformative as it is predicted to result in very early em- one scenario, a cancer cell, containing a disruption in the
bryonic lethality. Therefore, studies to examine the role of cyclin D/cdk4/pRb regulatory pathway, may no longer re-
CaM in the cell cycle in mammalian cells have focused on the quire Ca2⫹/CaM to regulate the activation of this pathway
use of antagonists of CaM function. Microinjection of mono- because it is already activated or unnecessary for G1 pro-
clonal antibodies against CaM inhibited DNA replication in gression in the tumor cell.
a dose-dependent manner (83). A series of pharmacological
inhibitors of CaM have been developed to probe its functions
in cells. W-7 and its derivatives (W-13) bind CaM and sub- D. In vivo studies of CaM function during cell growth
sequently, prevent activation of Ca2⫹/CaM-dependent tar- and proliferation
get enzymes. These compounds are cell permeable and dis-
Because most of the studies examining CaM in the cell
tribute through the cell (84). Treatment of a wide variety of
cycle have relied on manipulation of cultured cells, one
cells with W-7 or its derivatives inhibited proliferation (30,
would like to know whether some of the same findings are
55, 83– 86). Both W-7 and W-13 also prevented proliferation
true in a whole animal. One model system in which require-
and colony formation of breast cancer cell lines (87). When
ment of Ca2⫹/CaM in vivo has been tested is cardiomyocytes.
Chinese hamster ovary-K1 cells were released from plateau
Cardiomyocytes possess several distinct stages of cell pro-
phase, W-13 prevented cell cycle reentry. CaM appeared to
liferation. Normal cell division is limited to embryogenesis
be required at two points, early after mitogenic stimulation
in myocytes (92). In the perinatal period, myocytes continue
and late in G1 near the G1/S boundary (55). In human fi-
to undergo DNA synthesis in the absence of cytokinesis,
broblasts, addition of W-7 up to 8 h after mitogenic stimu-
resulting in polynucleate cells. After this process of
lation completely inhibited DNA synthesis, but if W-7 was
polynucleation, myocytes no longer synthesize DNA but
added later in G1 after pRb hyperphosphorylation, there was
often undergo hypertrophy in response to certain signals as
no inhibition of DNA synthesis (30, 33). It is intriguing that
those that occur in heart failure. As with most cells, the
both Ca2⫹ and CaM appear to be required at two points
proliferation of primary or secondary rat ventricular myo-
during reentry, early after mitogenic stimulation and in late
cytes required extracellular Ca2⫹, and chelation of extracel-
G1. Based on the work of Takuwa et al. (33), the point in late
lular Ca2⫹ resulted in a G1 arrest (93).
G1 when Ca2⫹/CaM is required is before the restriction point
To test the effects of CaM on myocyte proliferation, our
and pRb phosphorylation.
laboratory generated transgenic mice that overexpress CaM
Recently, the effects of W-13 on proteins that control pRb
in the heart using the atrial naturetic factor promoter. The
phosphorylation in G1, such as cyclin D/cdk4, have been
ventricles of these mice were both hypertrophic and hyper-
investigated in NRK cells. Although W-13 did not affect the
plasic (94). At birth, there was a 40% increase in the number
accumulation of cyclin D after mitogenic stimulation, it pre-
of ventricular myocytes over control animals (Colomer, J.,
vented the entry of cyclin D/cdk4 complexes into the nucleus
and A. R. Means, unpublished data). After birth, these mice
(85). Taules et al. isolated cyclin D/cdk4 complexes from
demonstrated increased DNA synthesis without cell division
NRK cells using CaM Sepharose and suggested that the
or polynucleation, resulting in increased polyploidy of the
interaction between CaM and cyclin D/cdk4 was mediated
nuclei. Finally, the myocytes overexpressing CaM became
by hsp90 and/or p21 (85, 88). However, how and if the
hypertrophic, and this hypertrophy receded as the CaM lev-
association between CaM and cyclin D-cdk4 regulates the
els fell. Therefore, CaM overexpression affected all stages of
nuclear import of cyclin D/cdk4 remains unknown. In any
myocyte proliferation and growth. However, the effect of
case, it is clear that Ca2⫹/CaM regulates pRb hyperphos-
CaM overexpression on myocyte proliferation is limited to,
phorylation in late G1, most likely via effects on cyclin
and does not extend beyond, the developmental period dur-
D/cdk4 (Fig. 1).
ing which myocyte numbers are expanded.
Interestingly, there are some striking similarities between
the studies of CaM function and cyclin D1 function in mam-
malian cells. Overexpression of both CaM and cyclin D1 E. Targets of CaM and cell proliferation
specifically accelerated the G1 phase of the cell cycle (60, 62,
89, 90). Inhibition of CaM or cyclin D1 function by antibody Ca2⫹/CaM bind and regulate numerous intracellular pro-
microinjection arrested cells in G1 (83, 91). One logical ex- teins involved in a myriad of pathways. Therefore, identi-
planation is that cyclin D1 and its cdk complexes are the fication of conserved Ca2⫹/CaM binding proteins that reg-
ultimate target of Ca2⫹/CaM-dependent cascades during G1 ulate cell cycle progression remains difficult. Genetic
progression. If the downstream target of Ca2⫹/CaM during systems, such as yeast and fungi, have proved essential in the
G1 is the cyclin D/cdk4/pRb pathway, it may explain some isolation of key cell cycle regulators, in particular cyclins and
of the changes in the Ca2⫹ requirement during transforma- cdks. Therefore, one could hypothesize that the Ca2⫹/CaM-
tion. One hypothesis is that a human cancer must subvert the dependent target proteins involved in cell cycle regulation
cyclin D/cdk4/pRb pathway by one of several methods: should be essential or, at the very least, loss of function
overexpression of cyclin D1 or cdk4, loss or mutation of cdk4 should result in growth defects in genetic model systems.
inhibitors (p15/p16 family), or loss of pRb function (13). However, whereas CaM is essential in yeast, fungi, and flies,

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only a handful of essential CaM binding proteins have been
identified.
Although CaM function has been extensively character-
ized in S. cerevisiae, this organism is unique relative to its
regulation of Ca2⫹ and function of CaM (95). Whereas CaM
from vertebrate systems binds four Ca2⫹ ions with each
EF-hand motif, CaM from S. cerevisiae binds only three Ca2⫹
ions, with the fourth EF-hand motif being nonfunctional for
Ca2⫹ binding (96). Although CaM is essential, high-affinity
Ca2⫹ binding to CaM is not essential because expression of
a mutant CaM, which does not bind Ca2⫹ with high affinity,
supported growth in budding yeast (97). Based on these
findings, it is not surprising that no essential Ca2⫹/CaM-
dependent target enzymes have been identified in S. cerevi-
siae (Table 1). Budding yeast does possess essential Ca2⫹-
independent CaM targets, such as the spindle pole body-
associated protein Nuf1p/Spc110p and the unconventional
(class II) myosin Myo2p (95). Previously, Nuf1p/Spc110p
has been classified as a Ca2⫹-independent CaM target be-
cause it was shown to interact with the mutant CaM, which
does not bind Ca2⫹ with high affinity in a yeast two-hybrid
assay (98). However, the CaM binding region of Nuf1p/
Spc110p possesses a Ca2⫹-dependent, not Ca2⫹-indepen-
dent, consensus sequence and using an in vitro CaM overlay
assay, CaM binding to Nuf1p/Spc110p was Ca2⫹-dependent
(99, 100). For a comprehensive discussion of CaM targets in
S. cerevisiae, we refer readers to a recent review by Cyert (95).
Therefore, studies in S. cerevisiae may be more useful in
understanding the Ca2⫹-independent functions rather than
the Ca2⫹-dependent functions of CaM.
In contrast to S. cerevisiae, S. pombe and A. nidulans required
high-affinity Ca2⫹ binding to CaM to support growth (101,
102). Although several Ca2⫹/CaM-dependent targets have
been isolated from S. pombe, these targets are either not es-
sential or have yet to be characterized (Table 1).
On the other hand, both Ca2⫹-dependent and Ca2⫹-inde-
pendent targets have been identified and shown to be es-
sential in A. nidulans. In terms of Ca2⫹-independent targets,
both an unconventional myosin, myosin A, and a Nuf1/
Spc110 homolog, An110, have been isolated from A. nidulans,
but only the myosin A gene has been silenced and demon-
strated to be essential (102, 103). In addition, A. nidulans
contains at least three genes that encode essential Ca2⫹/
CaM-dependent enzymes with homologs in mammalian
cells. These genes produce two Ca2⫹/CaM-dependent pro-
tein kinases (CMKA and CMKB) and the Ca2⫹/CaM-depen-
dent protein phosphatase 2B, calcineurin (104 –106).
Although yeast contains homologs of both calcineurin and
CaMK, neither calcineurin nor any CaMK is essential for
growth under normal conditions. However, null strains for
these genes either demonstrated mild growth defects or
impaired growth under certain environmental conditions.
Additionally, cultured mammalian cells show proliferative
defects when calcineurin or CaMK function is pharmaco-
logically inhibited. Therefore, we will focus on the possible
roles of calcineurin and CaMKs as downstream Ca2⫹/CaM
target enzymes that regulate proliferation in a variety of
circumstances.
IV. Role of Calcineurin in Cell Proliferation
A. Calcineurin structure and biochemistry
Calcineurin is a heterodimer composed of a catalytic sub-
unit, calcineurin A, and a Ca2⫹-binding regulatory subunit,
calcineurin B (107–110). The two subunits are tightly bound,
only being dissociated by denaturation, and both subunits
are essential for calcineurin function. Calcineurin A contains
an amino-terminal catalytic domain followed by the cal-
cineurin B binding domain and a regulatory domain (Fig. 3).
The regulatory domain contains a CaM binding region and
autoinhibitory domain. Binding of Ca2⫹/CaM to the regu-
latory domain leads to dramatic enzyme activation through
the relief of autoinhibition. The calcineurin B subunit consists
of four EF-hand Ca2⫹ binding motifs, similar to CaM, and a
conserved amino-terminal myristylation site.

TABLE 1. CaM targets in yeast and A. nidulans

Mammalian
Target Organism Essential CaM binding Function Ref.
homologs
2⫹ a
Nuf1p/Spc110p Kendrin S. cerevisiae Yes Ca -independent SPB regulation Reviewed in 95
Pcp1 Kendrin S. pombe Yes Ca2⫹-independenta SPB regulation 102
An110 Kendrin A. nidulans Unknown Ca2⫹-independenta Unknown 102
Myo2p Class V myosins S. cerevisiae Yes Ca2⫹-independent Polarized growth Reviewed in 95
MYOA Myosin I A. nidulans Yes Ca2⫹-independent Polarized growth 103
Secretion
Cna1p, Cna2p Calcineurin A S. cerevisiae No Ca2⫹-dependent Stress response Reviewed in 95
(Cnb1p) (calcineurin B) Ca2⫹ homeostasis
G2/M transition
Ppb1 Calcineurin A S. pombe No Ca2⫹-dependent Mating 119
Cell shape and polarity
CNA Calcineurin A A. nidulans Yes Ca2⫹-dependent G1 progression 106, 125
Cmk1p, Cmk2p CaMKII S. cerevisiae No Ca2⫹-dependent Stress response 163–165
Cmk1 CaMKI S. pombe No Ca2⫹-dependent Cell cycle 167
Cmk2 CaMK S. pombe No Ca2⫹-dependent Oxidative stress response 168, 183
CMKA CaMKII A. nidulans Yes Ca2⫹-dependent G2/M transition 105, 169
CMKB CaMKI A. nidulans Yes Ca2⫹-dependent Reentry from sporulation 104
CMKC CaMKK A. nidulans No Ca2⫹-dependent Reentry from sporulation 104
SPB, Spindle pole body.
a
Although Nuf1p/Spc110p has been previously characterized as a Ca2⫹-independent target of CaM based on two-hybrid studies, it has been
shown to bind CaM in a Ca2⫹-dependent manner in vitro, and Ca2⫹ has been implicated in its in vivo functions in S. cerevisiae (98 –100).

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 726 Endocrine Reviews, December 2003, 24(6):719 –736
FIG. 3. Schematic diagrams of calcineurin and CaMK. A, The cata-
lytic subunit of the serine/threonine protein phosphatase calcineurin,
calcineurin A, contains an amino-terminal catalytic domain followed
by a calcineurin B-binding domain (CnB), a CaM-binding domain
(CaM), and an autoinhibitory domain (AI). Calcineurin B contains
four EF-hand Ca2⫹ binding motifs and an amino-terminal myristy-
lation site. B, The multifunctional CaMKs contain an amino-terminal
catalytic domain followed by a regulatory domain containing the
overlapping CaM binding and autoinhibitory domains. CaMKII also
contains a carboxy-terminal association domain, and some isoforms
have an alternatively spliced NLS. CaMKI and CaMKIV do not con-
tain association domains but have homologous Thr phosphorylation
sites in their activation loops.
The Ca2⫹-dependent phosphatase activity is controlled by
both calcineurin B and CaM (107–110). Although calcineurin
remains inactive when the high-affinity Ca2⫹-binding site in
calcineurin B is occupied, Ca2⫹ binding to the low-affinity
sites enables some activation of the enzyme. However, ad-
dition of Ca2⫹/CaM results in a 10- to 100-fold activation due
to changes in Vmax. Importantly, calcineurin is activated after
small changes in intracellular Ca2⫹ concentration following
cell stimulation due to the highly cooperative nature of Ca2⫹
binding to CaM and the very high affinity of calcineurin for
Ca2⫹/CaM.
In mammalian systems, calcineurin is well known for its
role in T cell activation, and two clinically useful immuno-
suppressants, cyclosporin A and tacrolimus (FK506), prevent
calcineurin function. Calcineurin dephosphorylates the tran-
scription factor nuclear factor of activated T cells (NFAT),
allowing it to enter the nucleus and promote gene transcrip-
tion (111–113). However, calcineurin is not limited to T cells
but is found in all cell types. More recently, the function of
calcineurin in a wide variety of cells has been investigated.
B. Genetic analysis of calcineurin function
Although none of the three genes encoding calcineurin
subunits (CNA1, CNA2/CMP2, and CNB1) in S. cerevisiae
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Kahl and Means • Calcium/Calmodulin and the Cell Cycle
are essential, yeast calcineurin functions in the cellular
response to stress (95). When yeast cells were grown in
stressful environmental conditions, such as high ion con-
centrations or high temperature, the expression of cal-
cineurin-activated genes was induced. Similar to the reg-
ulation of NFAT, calcineurin dephosphorylated the
transcription factor Crz1p/Tcn1p, promoting its nuclear
accumulation and activity (114, 115). DNA microarray
analysis revealed that the induced genes are involved in
signaling pathways, ion and small molecule transport, cell
wall maintenance, and vesicular transport (116). There-
fore, calcineurin-deleted strains were sensitive to both
high pH and high osmolarity (95). Exposure to ␣-factor
also activated calcineurin-dependent gene expression, and
prolonged exposure to ␣-factor in the absence of cal-
cineurin function led to cell death (117). Additional func-
tions of calcineurin in S. cerevisiae include the regulation
of Ca2⫹ homeostasis and Swe1p activity at the G2/M tran-
sition (95).
In S. pombe, the catalytic subunit of calcineurin, ppb1, is not
essential, but null mutants displayed several defects (118,
119). First, the ppb1 null mutants showed impaired cytoki-
nesis at low temperature as well as defects in maintaining cell
shape and polarity. They also exhibited a mating defect and
were sterile. Interestingly, calcineurin mRNA expression
peaked during S phase and was induced under nitrogen-
starvation conditions (120). Although calcineurin is not es-
sential in yeast, the evidence suggests that it plays critical
roles in the response to stressful environmental conditions
and during mating.
The requirement for calcineurin for cell growth under
stressful environmental conditions has been recently
strengthened by studies in pathogenic fungi. Cryptococcus
neoformans causes meningoencephalitis in humans, par-
ticularly in immunocompromised hosts such as those with
AIDS. Although not essential for growth under normal
conditions, both the calcineurin A and B genes were re-
quired for growth at 37 C and for virulence (121–123).
Similar to yeast, calcineurin A was also required for mat-
ing in this fungi (124).
In contrast to yeast, in A. nidulans, the calcineurin A gene
is essential (106). To study the function of calcineurin in
this fungi, the endogenous calcineurin A gene was placed
under control of the conditional alcA promoter (125). In
repressing media, which reduced calcineurin expression,
germlings underwent only one round of DNA replication
and then arrested primarily in G1, with some cells arrested
at G2 and mitosis. These results suggested that calcineurin
was critical for G1 progression, but not for initial cell cycle
reentry from sporulation. However, there may have been
enough calcineurin present in the spores to allow passage
through the first G1 but then, as expression fell due to
protein turnover, cells were unable to transit through a
second G1. Interestingly, endogenous calcineurin mRNA
was maximally expressed at the G1/S boundary, suggest-
ing that it may be regulated in a cell cycle-dependent
manner. Therefore, calcineurin is crucial for G1 transition
in this filamentous fungi.
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle Endocrine Reviews, December 2003, 24(6):719 –736 727

C. Calcineurin function in mammalian systems
The importance of calcineurin in mammalian cells was
illuminated by the investigations into the immunosuppres-
sive actions of cyclosporin A. Cyclosporin A blocks the ac-
tivation and proliferation of quiescent T cells after T cell
receptor engagement (111–113). Calcineurin was identified
as the target of cyclosporin A. Its enzymatic activity was not
inhibited directly by cyclosporin A but rather by a complex
of cyclosporin A and cyclophilin, an intracellular prolyl
isomerase (126 –129). In T cells, Ca2⫹ transients lead to the
activation of calcineurin, which dephosphorylates NFAT
and leads to its nuclear import and subsequent transcrip-
tional induction of IL-2 (111). Therefore, calcineurin activity
is essential to T cell activation and reentry into the cell cycle
after T cell receptor engagement.
Although the T cell represents a very specialized system
of reentry from quiescence, cyclosporin A has antiprolifera-
tive effects in a wide variety of cells, including adenocarci-
noma cell lines, lymphoma and leukemia cell lines, keratin-
ocytes, fibroblasts, and smooth muscle cells (130 –134).
Where investigated, the cell cycle arrest induced by cyclo-
sporin A has been in G1, although distinct mechanisms have
been proposed (Table 2). For example, studies in both lym-
phoid and nonlymphoid cells have demonstrated that cy-
closporin A induced TGF␤ expression that, in turn, led to
increased levels of p21. This increase in p21 levels induced
a G1 arrest, and the effects of cyclosporin A were blocked
with neutralizing antibodies to TGF␤ (135, 136). Interest-
ingly, Tomono et al. (137) demonstrated that growth factor
stimulated Swiss 3T3 cells arrested in G1 with cyclosporin A
treatment and showed a reduction in cyclins A and E, but not
cyclin D1. Both of these studies suggest that cyclosporin A
inhibits G1 progression by preventing cdk2 activation.
In contrast, recent studies have implicated calcineurin
function earlier in G1 through the regulation of cyclin
D/cdk4 activation. These studies have implicated cal-
cineurin in the regulation of the transcription, and therefore
expression, of both cyclin D and cdk4. Cyclosporin A treat-
ment induced a G1 arrest in pancreatic acinar cells (AR42J)
with low levels of cyclin D protein (138). This reduction in
protein was associated with low levels of cyclin D1 mRNA,
and this transcriptional effect was mapped to the cAMP
response element present in the 5⬘-regulatory region of cyclin
D1. Interestingly, the steady-state levels of cAMP response
element binding protein were reduced in cyclosporin A-
treated cells, but how calcineurin regulated cAMP response
element binding protein levels was unclear. Next, calcineurin
and NFATc2 have been implicated in the regulation of the
cdk4 promoter (139). Evidence suggested that NFATc2 in-
hibits the basal activity of the cdk4 promoter; therefore, cells
lacking calcineurin A␣ or NFATc2 had higher levels of cdk4
protein. Taken together, these results suggest that cal-
cineurin function promotes cyclin D1 expression and inhibits
cdk4 expression. Because both cyclin D and cdk4 are tran-
scriptionally induced during G1, these results seem at odds.
However, one possibility is that the regulation of cyclin
D/cdk4 by calcineurin is cell type specific or is dependent on
the proliferative state of the cell.
In T lymphocyctes, Baksh et al. (140) demonstrated a more
direct relationship between calcineurin and cdk4. They
found that calcineurin could dephosphorylate and inactivate
cdk4 directly, suggesting that it may play a role in the in-

TABLE 2. Cell cycle effects of CaM antagonists, KN-93, cyclosporin A, and FK506 in mammalian cells

Inhibitor Target Cell line Results Ref.

W-7 CaM CHO-K1 Inhibits proliferation 84
W-13 CaM CHO-K1 Arrests at two points upon release from plateau phase: 55
Early in G0/G1 and near G1/S transition
W-7 CaM WI-38 Arrests in G1 after mitogenic stimulation: 30, 33
IMR-90 Low histone H1 activity
Hypophosphorylated Rb
W-13 CaM NRK Arrests in G1 after mitogenic stimulation: 85
Low cyclin D/cdk4 activity
Cyclin D/cdk4 remains cytoplasmic
W-7, W-13, TFP CaM MDA-MB-231 Inhibits colony formation 87
KN-93 CaMK HeLa Arrests in G1: 170
Elevated histone H1 activity
KN-93 CaMK NIH 3T3 Arrests in G1 after mitogenic stimulation: 171, 172
Decreased cdk4 activity and decreased cyclin D1
Decreased cdk2 activity and elevated p27
KN-93 CaMK HeLa Arrests at G2/M: 175
Prevents cdc25c phosphorylation and activation
CsA/FK506 CNA A541 Inhibits DNA synthesis after mitogenic stimulation 133
CsA/FK506 CNA Swiss 3T3 Inhibits DNA synthesis after mitogenic stimulation: 134, 137
Decreased cyclins E and A
CsA CNA T cells Inhibition of cell cycle progression: 136
A-549 Induction of p21 via TGF␤
CsA CNA Keratinocytes Inhibits proliferation and DNA synthesis 130
CsA CNA Smooth muscle cells Inhibits DNA synthesis after mitogenic stimulation 132
Dermal fibroblasts
CsA/FK506 CNA Jurkat cells Increased cdk4 activity 140
CsA CNA AR42J Decreased cyclin D1 mRNA and protein 138
CsA/FK506 CNA Lymphocytes Increased cdk4 expression 139
CsA, Cyclosporin A; CNA, calcineurin A; CHO, Chinese hamster ovary.

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 728 Endocrine Reviews, December 2003, 24(6):719 –736
activation of cdk4 in mitosis (140). In our laboratory, we have
examined the requirement for calcineurin in normal human
fibroblasts (WI-38). We chose diploid fibroblasts, which are
strictly dependent on Ca2⫹/CaM for proliferation, because
they are unlikely to possess mutations or alterations in key
cell cycle-regulatory pathways. Similar to Schneider et al.
(138), we found that cyclosporin A induces a G1 arrest that
is characterized by low levels of cyclin D1 protein (our un-
published data). In contrast to their results in pancreatic
acinar cells, we found normal levels of cyclin D1 mRNA). In
WI-38 cells, cyclosporin A dramatically reduced the amount
of newly synthesized cyclin D1 protein, and expression of
constitutively active calcineurin promoted cyclin D1 synthe-
sis during mid-G1.
Clearly, the regulation of cyclin D/cdk4 by calcineurin
becomes a complicated issue with calcineurin potentially
regulating the transcription and translation of cyclin D1 as
well as the transcription and phosphorylation status of cdk4.
Although cyclosporin A prevents G1 progression in several
cell types, the mechanisms by which it induces a cell cycle
arrest are diverse; therefore, one wonders whether these
effects are related or merely distinct pathways in each cell
type.
Although it is clear that cyclosporin A possesses antipro-
liferative effects in numerous cell types, what is not clear is
whether these effects are specifically due to calcineurin in-
hibition. Both cyclosporin A and FK506 bind cyclophilins
and FK506 binding proteins (FKBPs) respectively (collec-
tively known as immunophilins), and the resulting drug-
immunophilin complexes inhibit both the Ca2⫹ and Ca2⫹/
CaM-stimulated activity of calcineurin (128). The
antiproliferative effects of cyclosporin A require higher drug
concentrations than does inhibition of T cell activation, and
FK506 is less potent than cyclosporin A in its antiproliferative
effects, although FK506 is a more potent immunosuppressive
agent (141, 142). Therefore, the question arises whether cal-
cineurin, immunophilins, or both are the targets of cyclo-
sporin A and FK506 in cells other than T cells.
Although both immunosuppressive compounds inhibit
the prolyl isomerase activity of immunophilins, in T cells, the
concentration of immunophilins far exceeds that of cal-
cineurin and, as a result, the low drug concentrations used
for immunosuppression inhibit the majority of calcineurin
while binding a relatively small fraction of the total pool of
immunophilins. Other cells, such as neurons and cardiomy-
ocytes, have approximately 40 times more calcineurin than
T cells and, therefore, higher drug concentrations should be
required to inhibit the majority of calcineurin and a greater
fraction of the immunophilin pool is bound to drug and
therefore inhibited (141, 142). Additionally, only some forms
of cyclophilins and FKBPs are able to form active drug-
immunophilin complexes capable of inhibiting calcineurin
(143). In most cases, the cellular complement of cyclophilins
and FKBPs is unknown and the active pool may be limited.
Evidence suggests that immunophilins may limit the degree
of calcineurin inhibition. At high concentrations, these drugs
are unable to inhibit all calcineurin activity, and cyclosporin
A inhibits a greater percentage of calcineurin than FK506.
Addition of exogenous immunophilins increases the degree
of calcineurin inhibition, suggesting that immunophilins
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Kahl and Means • Calcium/Calmodulin and the Cell Cycle
may be limiting for calcineurin inhibition (144). This idea is
supported by the fact that transfection of T cells with cyclo-
philins A or B as well as FKBP12 renders the cells more
sensitive to cyclosporin A and FK506, respectively (143). In
T cells, 100% calcineurin inhibition is not required to fully
inhibit NFAT dephosphorylation, but 100% calcineurin in-
hibition may be necessary in other cellular contexts (144).
Importantly, examples exist for both cyclosporin A and
FK506 having cellular effects that are independent of cal-
cineurin inhibition (110, 142). One must realize that inhibi-
tion or disruption of immunophilins, or other unknown pro-
teins, may contribute to effects seen with cyclosporin A and
FK506. Although cyclosporin A and FK506 represent the best
pharmacological inhibitors for probing calcineurin function
in cells and animals, using these agents does not guarantee
that any results are due solely to disruption of calcineurin
function.
V. Role of Ca2ⴙ/CaM-Dependent Kinases (CaMKs) in
Cell Proliferation
A. Structure and biochemistry of CaMKs
The multifunctional CaMKs are a family of serine/threo-
nine protein kinases that include CaMKI, CaMKII, and
CaMKIV (145–147). These kinases have an amino-terminal
catalytic domain followed by a carboxy-terminal regulatory
domain (Fig. 3). The regulatory domain consists of overlap-
ping autoinhibitory and Ca2⫹/CaM binding domains. Sim-
ilar to calcineurin, the autoinhibition is relieved upon Ca2⫹/
CaM binding. CaMKII, unlike CaMKI and CaMKIV, has an
additional association domain carboxy terminal from the
regulatory domain (148 –150). This domain enables CaMKII
to form multimeric structures, whereas CaMKI and CaMKIV
are monomeric enzymes (145, 147). Although all these ki-
nases are regulated by phosphorylation, the mechanism and
enzymatic consequences differ between the kinases (Table 3).
After Ca2⫹/CaM binding, CaMKII autophosphorylates in an
intraholoenzyme, intersubunit reaction (148 –150). Phos-
phorylation of Thr286 has two important consequences.
First, the enzyme becomes autonomous, or Ca2⫹/CaM in-
dependent, after the dissociation of Ca2⫹/CaM. Second, the
enzyme acquires a property called “CaM-trapping,” in
which the affinity of the enzyme for CaM is increased more
than 1000-fold. Recently, these changes in CaMKII after
autophosphorylation have been shown to be mimicked
by CaMKII association with the N-methyl-d-aspartate
receptor NR2B subunit in the absence of phosphorylation
(151). In contrast to CaMKII, both CaMKI and CaMKIV are
phosphorylated on an activation-loop threonine by an up-
stream kinase, Ca2⫹/CaM-dependent protein kinase kinase
(CaMKK) (147, 152). This phosphorylation results in maxi-
mal enzyme activation. For CaMKIV, phosphorylation
allows a considerable degree of autonomous activity. How-
ever, recent work on CaMKI suggests that this phosphory-
lation may not strictly regulate enzyme activity. Rather, the
peptide substrate specificity changes between the dephos-
phorylated and phosphorylated enzyme, resulting in acti-
vation-independent and -dependent peptide substrates
(153). Although this idea is provocative, the identification of
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle
TABLE 3. Properties of the mammalian, multifunctional CaMKs
CaMKI CaMKIV
Tissue distribution Ubiquitous Limited (testis, thymus,
and brain)
Subcellular Cytoplasmic Predominantly nuclear
localization
Subunit composition Monomeric Monomeric
Phosphorylation Thr177 phosphorylation by Thr196 phosphorylation
CaMKK by CaMKK and
autophosphorylation
Autonomous activity None Partial
Inhibition by KN-62 0.8 ␮M 3 ␮M
(KI)
Substrate consensus Hyd-X-R-X-X-S/T-X-X-X-Hyd Hyd-X-R-X-X-S/T
sequence
endogenous protein substrates that are activation indepen-
dent or -dependent remains an important challenge.
Another distinction between the multifunctional CaMKs is
tissue expression and subcellular distribution. There are four
genes encoding multiple isoforms of CaMKII. Two genes,
CaMKII␣ and CaMKII␤, are expressed predominantly in
neurological tissues, and two genes, CaMKII␦ and CaMKII␥,
are expressed predominantly in somatic tissues (145, 150).
Additionally, these CaMKII gene products are processed into
numerous splice variants that are differentially expressed in
tissues. Although most splice variants of CaMKII are cyto-
plasmic enzymes, an alternatively spliced NLS enables at
least one isoform encoded by each of the four genes to be a
predominantly nuclear enzyme (145). Interestingly, tumor
cells expressed different variants of CaMKII, although the
functional relevance of changes in CaMKII variants during
tumorigenesis is unknown (154, 155).
In terms of protein structure and enzymatic activation,
CaMKI and CaMKIV are fairly similar. However, these en-
zymes distinguish themselves by tissue distribution and sub-
cellular localization as well as by substrate preference (147).
CaMKI is ubiquitously expressed and localized to the cyto-
plasm. In contrast, CaMKIV expression is more limited, with
the highest levels of protein found in brain, thymus, and
testis. This enzyme is largely nuclear. Due to its limited
distribution, CaMKIV is unlikely to be universally required
for proliferation. However, CaMKIV expression is up-regu-
lated in several types of tumors, including lung, endometrial,
and ovarian cancers (156 –158). Whereas CaMKI and
CaMKIV have similar substrate preferences based on peptide
studies with the consensus sequence Hyd-X-R-X-X-S/T, our
laboratory has recently demonstrated that CaMKI and
CaMKIV phosphorylate different sites within an amino-
terminal fragment of p300 (159). CaMKI phosphorylated
Ser89 [84LLRSGSSPNL (93)], which corresponds to the ex-
pected consensus sequence, whereas CaMKIV phosphory-
lated Ser24 [19SSPALSASAS (28)], which represents a novel
site with little similarity to the proposed CaMKIV consensus
site based on peptide phosphorylation studies.
In mammalian cells, the CaMKK enzymes, which phos-
phorylate CaMKI and CaMKIV, consist of two enzymes,
CaMKK␣ and CaMKK␤ (147, 152). These enzymes share a
similar structure to other CaMKs with an amino-terminal
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CaMKII Ref.
Ubiquitous Reviewed in 145, 147
CaMKII␣/␤–neuronal
CaMKII␦/␥–somatic
Cytoplasmic and nuclear (if NLS Reviewed in 145, 147
present)
Homo- or heteromultimeric Reviewed in 145, 147
Intrasubunit autophosphorylation Reviewed in 145, 147
on Thr286
Yes via autophosphorylation or Reviewed in 145, 147
NR2 binding
0.8 ␮M 184
Hyd-X-R-NB-X-S/T 185, 186
catalytic domain and a carboxy-terminal regulatory domain.
Both kinases phosphorylate and activate CaMKI and
CaMKIV equally well. At the current time, the major differ-
ence shown to exist between the two forms is the degree of
Ca2⫹/CaM dependence. Although CaMKK␣ was dependent
on Ca2⫹/CaM binding to relieve autoinhibition, CaMKK␤
possessed significant activity in the absence of Ca2⫹/CaM
(160 –162). CaMKK␣ and CaMKK␤ are highly expressed in
neurological tissues with variable expression in other tissues.
Interestingly, all cells that expressed CaMKIV also expressed
CaMKK␤, leading to speculation that CaMKI and CaMKIV
might be regulated by different CaMKKs in the cell (160).
However, both kinases were localized to the cytoplasm.
Therefore, a major dilemma in the field arises: how does a
cytoplasmic CaMKK phosphorylate the nuclear enzyme,
CaMKIV, or could there be other nuclear kinases capable of
phosphorylating CaMKIV?
B. Genetic analysis of CaMK function
In S. cerevisiae, two genes homologous to CaMKII, cmk1
and cmk2, have been identified (163–165). Neither gene is
essential when deleted alone or in combination. However,
deletion of cmk1 and cmk2 lowered the LD50 of pheromone,
suggesting that both calcineurin and CaMK have a role in the
response of yeast cells to pheromone (166). In S. pombe, two
CaMK genes have also been identified. Unfortunately, the
effects of deleting cmk1, a CaMKI homolog, has yet to be
characterized in fission yeast. However, its mRNA expres-
sion was regulated in a cell cycle-dependent manner, peak-
ing at G1/S (167). A second CaMK homologous gene, cmk2,
was not essential, but its mRNA also peaked at G1/S (168).
Therefore, examination of the effects of cmk1 deletion, either
alone or in combination with cmk2, on S. pombe growth will
be informative.
In A. nidulans, three CaMK homologs have been identified:
CMKA, a CaMKII homolog; CMKB, a CaMKI or CaMKIV
homolog; and, CMKC, a CaMKK homolog (104, 105, 169). In
contrast to yeast, two of these genes are essential in A. nidu-
lans. CmkA was essential and required for the G2/M transi-
tion (105). When constitutively active CMKA was overex-
pressed, spores failed to enter the first S phase and
prematurely activated NIMXcdc2. CmkB was also essential but
 730 Endocrine Reviews, December 2003, 24(6):719 –736
appeared to be required sometime between sporulation and
initial S phase entry. Strains with delayed and reduced
CMKB expression demonstrated a delay in reentry from
sporulation as well as in NIMXcdc2 activation as demon-
strated by histone H1 phosphorylation assays (104). Al-
though not essential, cmkC null strains also showed a delay
in NIMXcdc2 activation after sporulation. These results sug-
gested that the CaMKI homolog, CMKB (and perhaps its
upstream activating kinase, CMKC), regulated NIMXcdc2 ac-
tivation in late G1 before DNA replication, and that the
CaMKII homolog, CMKA, regulated the G2/M transition.
C. CaMK function in mammalian systems
Similar to the findings in A. nidulans, studies in mamma-
lian cells suggest that one or more CaMK functions in G1 as
well as at the G2/M transition. One drawback to these studies
is that the majority of them relied on the use of the selective
CaMK inhibitors, KN-62 or KN-93. Although often sug-
gested to be CaMKII specific, KN-62 actually inhibits all three
multifunctional CaMKs with similar efficacy (Table 3).
KN-62 or KN-93 demonstrated antiproliferative effects in a
variety of cells. In HeLa cells, KN-93 caused a G1 arrest (170).
At the arrest point, p13-precipitable histone H1 kinase ac-
tivity was elevated 4-fold, suggesting the arrest was down-
stream of cdk2 activation. However, the activities of neither
cyclin E/cdk2 nor cyclin A/cdk2 were evaluated individu-
ally. In NIH 3T3 cells, KN-93 arrested both asynchronous
cells and mitogen-stimulated cells in G1 (171). With asyn-
chronous cells, the arrest was reversible after 2 d of drug
treatment, but by 3 d of treatment, the cells began to undergo
apoptosis. In subsequent experiments, KN-93 treatment was
found to prevent cdk4 and cdk2 activation as well as pRb
phosphorylation (172). The authors concluded that cdk4 was
inactive due to reduced levels of cyclin D, although cyclins
E and A were expressed normally. The reduction of cdk2
activity was associated with elevated levels of p27, but the
amount of p27 associated with cdk complexes was not eval-
uated. These results in NIH 3T3 cells differ dramatically from
the results in HeLa cells in which cdk activity was elevated.
One way to rationalize these results is to speculate that
KN-93 may arrest these cells at different points in G1 with
NIH 3T3 cells arresting at the first CaM-dependent step in G1
early after mitogenic stimulation and HeLa cells arresting
later in G1 at the second CaM-dependent step near the G1/S
boundary. Another possibility is that HeLa, a transformed
cell line, has lost a CaM-dependent step in G1 which is still
present in NIH 3T3 cells, which are immortalized but not
transformed.
To learn more about the function of CaMKs in G1 and to
identify which CaMK may be required for G1 progression,
we examined the requirements for CaMK function in G1
progression in normal fibroblasts (WI-38). In these cells,
KN-93 treatment arrested cells in G1 with low levels of cdk4
activity and hypophosphorylated pRb (our unpublished re-
sults). Unlike results of Morris et al. (172) in which cyclin D
levels were reduced by KN-93 treatment, cyclin D was ex-
pressed at normal levels in WI-38 cells treated with KN-93.
Indeed, cyclin D/cdk4 kinase complexes were assembled,
phosphorylated, and localized to the nucleus in KN-93-
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Kahl and Means • Calcium/Calmodulin and the Cell Cycle
treated cells (Fig. 2). Therefore, CaMK inhibition appeared to
block a very late, uncharacterized step in cdk4 activation,
suggesting that a CaMK may be the late G1 target of Ca2⫹/
CaM. To evaluate the relevant CaMK, we expressed mutant
forms of the two multifunctional CaMKs expressed in WI-38
cells (CaMKI and CaMKII). These kinase-deficient enzymes
have the lysine in the ATP-binding loop mutated to prevent
ATP binding. We asked whether overexpression of either
protein could act as a “dominant negative” by mimicking the
KN-93 arrest. Expression of kinase-deficient CaMKI, but not
CaMKII, during G1 prevented cdk4 activation similar to KN-
93. As with the studies in A. nidulans, these results suggest
that CaMKI, rather than CaMKII, regulates G1 progression.
One potential role for CaMKII during G1/S is centrosome
duplication. In 1990, CaMKII was localized to the centrosome
in mammalian cells (173). Recently, Matsumoto and Maller
(174) implicated CaMKII function in centrosome duplication
in Xenopus egg extracts. Both Ca2⫹ chelation and CaMKII
inhibition blocked centrosome duplication in egg extracts.
Importantly, readdition of CaMKII and CaM to the extracts
restored centrosome duplication in this system. To date, the
function of CaMKII in regulating centrosome duplication in
mammalian cells is unknown, but it will be critical to further
investigate this pathway because many tumors show excess
numbers of centrosomes due to aberrant duplication.
Whether or not CaMKII is essential for G1 progression, this
enzyme probably acts to regulate the G2/M transition and
mitotic progression in mammalian systems. When HeLa cells
were synchronized in S phase and released into KN-93, they
arrested at G2/M without detectable cdc25C hyperphospho-
rylation (175). At the G2/M transition, cdc25C, a dual-spec-
ificity phosphatase, is activated by phosphorylation. Once
activated, it removes the inhibitory tyrosine phosphorylation
on cdc2, leading to its activation (176, 177). In vitro, CaMKII
phosphorylated inactive cdc25C and marginally increased its
activity, suggesting that CaMKII may be one relevant cdc25C
kinase in cells (175). Again, these results differ dramatically
from earlier results in which HeLa cells arrested in G1, not in
G2, upon KN-93 treatment. Another group found that KN-93
treatment delayed mitotic progression in HeLa cells, but
those cells did progress through mitosis (178). Although all
these groups used HeLa cells, the major difference between
the studies was the proliferative state of cells when KN-93
was added, one being asynchronously growing cells and the
other being cells synchronized in S phase. One explanation
could be that cells in mid-G1 contain about 50% less CaM
than in S phase and, therefore, the cells are more sensitive to
KN-93 in G1 (55, 56). By using an S phase synchronization,
the G1 arrest point was avoided, which enabled evaluation
of the G2/M transition. However, a role for CaMKII in G2/M
progression is supported by the work in A. nidulans, in which
the CaMKII homolog was required for this transition, and
expression of constitutively active CaMKII prematurely ac-
tivated NIMXcdc2 (105, 179).
CaMKII is clearly the target of the Ca2⫹ signal required for
the metaphase to anaphase transition. Unfertilized marine
eggs are arrested at metaphase of meiosis II by cytostatic
factor, the product of the c-mos protooncogene. Upon fertil-
ization, intracellular Ca2⫹ increases result in the inactivation
of cytostatic factor and M phase-promoting factor, the
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle Endocrine Reviews, December 2003, 24(6):719 –736 731

cyclin/cdc2 complex (180 –182). First, it was demonstrated
that CaM was required for cyclin degradation, followed by
the demonstration that CaMKII was the target of CaM at this
point. Although inhibitors of CaMKII blocked cyclin degra-
dation and cdc2 inactivation after a Ca2⫹ signal, constitu-
tively active CaMKII promoted cyclin degradation and cdc2
inactivation in the absence of a Ca2⫹ signal. Therefore,
CaMKII is a critical regulator of the metaphase to anaphase
transition in egg extracts, but this pathway still needs to be
investigated during a mitotic, rather than meiotic, cell cycle.
VI. Conclusions and Perspectives
In summary, cells require Ca2⫹/CaM-dependent path-
ways to grow and divide. Although Ca2⫹/CaM-dependent
pathways probably act at innumerable points during cell
cycle progression, the best-characterized pathways to date
are during G1 and G2/M progression. Unfortunately, con-
served Ca2⫹/CaM-dependent pathways common to both
unicellular and multicellular eukaryotes that regulate pro-
gression through these cell cycle transitions remain ambig-
uous. Although not absolutely required for all types of cell
proliferation, calcineurin and the family of CaMKs represent
two potential Ca2⫹/CaM-dependent enzymes that regulate
cell cycle transitions. Importantly, experimental evidence
demonstrates that these homologous enzymes regulate sim-
ilar cell cycle transitions in A. nidulans and mammalian cells.
Both Ca2⫹ and CaM are required at least two points in G1,
and both points are prior to the activation of cyclin D/cdk4
and pRb hyperphosphorylation (Fig. 4). Available evidence
strongly suggests that these Ca2⫹/CaM-dependent path-
ways directly or indirectly regulate cyclin D1/cdk4 activity.
Cyclosporin A, an inhibitor of calcineurin function, regulates
the expression of both cyclin D1 and cdk4. Intriguingly,
cyclosporin A appears to inhibit cyclin D1 accumulation,
whereas it appears to promote cdk4 expression. At first, these
results seem incompatible, but the studies were carried out
by different groups in different cell types. Additional exper-
iments will be necessary to determine whether calcineurin
plays a more general role in regulating cyclin D1/cdk4 ex-
pression in multiple tissues and cell types. At this point, we
favor a function of calcineurin in promoting cyclin D1 ac-
cumulation in early G1, which would be consistent with the
early G1 requirement for Ca2⫹/CaM.
One or more of the CaMKs must also be required for G1
progression because KN-93, an inhibitor of the multifunc-
tional CaMKs, prevents G1 progression in both normal and
transformed cells (Fig. 4). Whereas the nature of this arrest

FIG. 4. Schematic model of how calcineurin and CaMKs regulate mammalian cell cycle transitions. In early/mid G1, inhibitors of both
calcineurin and CaMK arrest cells before the activation of cyclin D1/cdk4. Cyclosporin A, an inhibitor of calcineurin function, arrests cells with
low levels of cyclin D1 protein, which may be due to transcriptional or translation effects or both. Calcineurin also appears to regulate cdk4
transcription as well as the phosphorylation status of cdk4 in certain cell types. KN-93, an inhibitor of the multifunctional CaMKs, also arrests
cells before cyclin D1/cdk4 activation, but the nature of this G1 arrest varies between cell types. Some studies suggest an early G1 arrest with
low levels of cyclin D1 protein, whereas others suggest a much later arrest after cyclin D1/cdk4 complex assembly. In late G1 or S phase, inhibition
of calcineurin or CaMK leads to p21 and p27 accumulation, respectively. Increased levels of p21 or p27 are sufficient to lead to cdk2 inactivation
and a cell cycle block. Finally, CaMKII is the most likely target of Ca2⫹/CaM-dependent pathways at the G2/M and anaphase to metaphase
transitions. Inhibition or loss of CaMKII function leads to a G2 arrest with inactive cdc2 in both mammalian and fungal systems.

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 732 Endocrine Reviews, December 2003, 24(6):719 –736
varies between cell types, KN-93 arrests nontransformed
cells before cyclin D1/cdk4 activation. Unfortunately, the
mechanism by which CaMK inhibition prevents cdk4 acti-
vation differs among cell types, and no previous studies have
addressed which CaMK is required at this point in G1. We
found that kinase-deficient CaMKI, but not CaMKII, acts like
a “dominant negative,” and its expression arrested cells with
low cdk4 activity and hypophosphorylated pRb. Could
CaMKI, not CaMKII, be the target of Ca2⫹/CaM-dependent
pathways in G1? Studies in A. nidulans support a role for
CaMKI function during G1. CMKA, the CaMKI homolog, is
essential and reduction of its expression delayed the activa-
tion of NIMXcdc2 after sporulation and before DNA synthe-
sis. Taken together, CaMKI probably represents one target of
Ca2⫹/CaM-dependent signaling during G1 progression and
acts to regulate the activation of the G1 cdks.
Some reports implicate both calcineurin and CaMK in the
progression through late G1 and S phase. Cyclosporin A
results in p21 accumulation secondary to TGF␤ induction,
and KN-93 causes a rise in p27 levels. An increase in either
p21 or p27 is sufficient to inhibit cdk2 complexes in late G1
or S phase.
One problem that arises from these studies is the fact that
it becomes unclear whether the accumulation of p21/p27
proteins represents a “direct” target or is merely the cellular
response to a prolonged arrest earlier in G1.
Currently, all the experimental evidence in mammalian
cells, A. nidulans, and Xenopus extracts points to CaMKII and
its homologs as the target of Ca2⫹/CaM-dependent path-
ways at the G2/M and metaphase to anaphase transitions
(Fig. 4). In HeLa cells, KN-93 blocked G2 progression with
low cdc2 activity. In vitro, CaMKII directly phosphorylated
and increased the activity of cdc25C, the phosphatase re-
sponsible for cdc2 activation at the G2/M transition. In A.
nidulans, CMKA, the CaMKII homolog, is essential and re-
quired for G2/M progression. Overexpression of a constitu-
tively active CMKA prematurely activated NIMXcdc2, sug-
gesting that CMKA may also phosphorylate and activate
NIMTcdc25 in this organism. Therefore, at the G2/M tran-
sition, Ca2⫹/CaM regulates CaMKII, which may promote
cdc2 activation.
Finally, CaMKII is the target of Ca2⫹/CaM at the meta-
phase to anaphase transition during a meiotic cell cycle. In
Xenopus egg extracts, CaMKII clearly regulates the meta-
phase to anaphase transition, and its activity led to the deg-
radation of cyclin and inactivation of cdc2. Unfortunately,
this pathway has yet to be characterized in cultured mam-
malian cells. The fact that KN-93 delayed the metaphase to
anaphase transition in HeLa cells suggests that CaMKII may
play a role in the regulation of this transition in a mitotic cell
cycle, but is not absolutely required.
Hormones, growth factors, and cytokines act as critical
regulators of cell cycle progression. Both steroid and peptide
hormones are known to regulate the activity of cyclin/cdk
complexes. Hormones function at multiple levels of cdk reg-
ulation including the expression of cyclins and CKIs, the
subcellular localization of cyclin/cdk complexes, and the
subunit composition of cdk complexes. Hormones and
growth factors elicit cellular responses by inducing intracel-
lular Ca2⫹ transients. Ca2⫹ and its intracellular receptor,
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Kahl and Means • Calcium/Calmodulin and the Cell Cycle
CaM, are universally required for cell cycle progression.
Based on our current understanding, Ca2⫹/CaM-dependent
pathways are also involved at multiple levels of cdk regu-
lation, similar to hormone action. Future experimental stud-
ies designed for understanding how hormones affect the
Ca2⫹/CaM-dependent pathways involved in cell cycle pro-
gression are greatly needed as well as an understanding of
how these pathways differ in various tissues and cell types
within those tissues. Additionally, both hormonal regulation
and Ca2⫹/CaM-dependent regulation of cell proliferation
are often disrupted in human tumors. By investigating how
hormones and Ca2⫹/CaM regulate normal cell proliferation
in different tissues, we ultimately may gain insight into how
these pathways are disrupted in human cancer.
Acknowledgments
We thank James Joseph for valuable discussions and critical reading
of the manuscript. We also thank Josep Colomer for discussions regard-
ing his unpublished data and Libby MacDougall for technical assistance.
Address all correspondence and requests for reprints to: Dr. Anthony
R. Means, Department of Pharmacology and Cancer Biology, Box 3813,
Duke University Medical Center, Durham, North Carolina 27710. E-
mail: means001@mc.duke.edu
This work was supported by NIH Grants HD-07503 and GM-33976
(to A.R.M.) and by an NIH Medical Scientist Training Program award
(to C.R.K.).
References
1. Berridge MJ, Lipp P, Bootman MD 2000 The versatility and uni-
versality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21
2. Carafoli E 2002 Calcium signaling: a tale for all seasons. Proc Natl
Acad Sci USA 99:1115–1122
3. Petersen OH, Petersen CC, Kasai H 1994 Calcium and hormone
action. Annu Rev Physiol 56:297–319
4. Taylor CW 1995 Why do hormones stimulate Ca2⫹ mobilization?
Biochem Soc Trans 23:637– 642
5. Hanahan D, Weinberg RA 2000 The hallmarks of cancer. Cell
100:57–70
6. Elledge SJ 1996 Cell cycle checkpoints: preventing an identity
crisis. Science 274:1664 –1672
7. Nigg EA 2001 Mitotic kinases as regulators of cell division and its
checkpoints. Nat Rev Mol Cell Biol 2:21–32
8. Morgan DO 1995 Principles of CDK regulation. Nature 374:
131–134
9. Morgan DO 1997 Cyclin-dependent kinases: engines, clocks, and
microprocessors. Annu Rev Cell Dev Biol 13:261–291
10. Morgan DO, Fisher RP, Espinoza FH, Farrell A, Nourse J, Cham-
berlin H, Jin P 1998 Control of eukaryotic cell cycle progression by
phosphorylation of cyclin-dependent kinases. Cancer J Sci Am
4(Suppl 1):S77–S83
11. Lew DJ, Weinert T, Pringle JR 1997 Cell cycle control in Saccha-
romyces cerevisiae. In: Pringle JR, Broach JR, Jones EW, eds. The yeast
Saccharomyces: cell cycle and cell biology. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press; 607– 695
12. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative
regulators of G1-phase progression. Genes Dev 13:1501–1512
13. Sherr CJ 1996 Cancer cell cycles. Science 274:1672–1677
14. Ekholm SV, Reed SI 2000 Regulation of G1 cyclin-dependent ki-
nases in the mammalian cell cycle. Curr Opin Cell Biol 12:676 – 684
15. Lundberg AS, Weinberg RA 1998 Functional inactivation of the
retinoblastoma protein requires sequential modification by at least
two distinct cyclin-cdk complexes. Mol Cell Biol 18:753–761
16. Weinberg RA 1995 The retinoblastoma protein and cell cycle con-
trol. Cell 81:323–330
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle
17. Nevins JR, DeGregori J, Jakoi L, Leone G 1997 Functional analysis
of E2F transcription factor. Methods Enzymol 283:205–219
18. Nevins JR, Leone G, DeGregori J, Jakoi L 1997 Role of the Rb/E2F
pathway in cell growth control. J Cell Physiol 173:233–236
19. Yam CH, Fung TK, Poon RY 2002 Cyclin A in cell cycle control and
cancer. Cell Mol Life Sci 59:1317–1326
20. Stillman B 1996 Cell cycle control of DNA replication. Science
274:1659 –1664
21. Kishimoto T, Okumura E 1997 In vivo regulation of the entry into
M-phase: initial activation and nuclear translocation of cyclin
B/Cdc2. Prog Cell Cycle Res 3:241–249
22. Ortega S, Malumbres M, Barbacid M 2002 Cyclin D-dependent
kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602:
73– 87
23. Hall M, Peters G 1996 Genetic alterations of cyclins, cyclin-depen-
dent kinases, and Cdk inhibitors in human cancer. Adv Cancer Res
68:67–108
24. Pestell RG, Albanese C, Reutens AT, Segall JE, Lee RJ, Arnold A
1999 The cyclins and cyclin-dependent kinase inhibitors in hor-
monal regulation of proliferation and differentiation. Endocr Rev
20:501–534
25. Hickie RA, Wei JW, Blyth LM, Wong DY, Klaassen DJ 1983
Cations and calmodulin in normal and neoplastic cell growth reg-
ulation. Can J Biochem Cell Biol 61:934 –941
26. Hazelton B, Mitchell B, Tupper J 1979 Calcium, magnesium, and
growth control in the WI-38 human fibroblast cell. J Cell Biol 83:
487– 498
27. Whitfield JF, Boynton AL, MacManus JP, Sikorska M, Tsang BK
1979 The regulation of cell proliferation by calcium and cyclic AMP.
Mol Cell Biochem 27:155–179
28. Boynton AL, Whitfield JF, Isaacs RJ 1976 The different roles of
serum and calcium in the control of proliferation of BALB/c 3T3
mouse cells. In Vitro 12:120 –123
29. Boynton AL, Whitfield JF, Isaacs RJ, Tremblay R 1977 The control
of human WI-38 cell proliferation by extracellular calcium and its
elimination by SV-40 virus-induced proliferative transformation.
J Cell Physiol 92:241–247
30. Takuwa N, Zhou W, Kumada M, Takuwa Y 1992 Ca2⫹/calmod-
ulin is involved in growth factor-induced retinoblastoma gene
product phosphorylation in human vascular endothelial cells.
FEBS Lett 306:173–175
31. Boynton AL 1988 Calcium and epithelial cell proliferation. Miner
Electrolyte Metab 14:86 –94
32. Whitfield JF 1992 Calcium signals and cancer. Crit Rev Oncog
3:55–90
33. Takuwa N, Zhou W, Kumada M, Takuwa Y 1993 Ca2⫹-dependent
stimulation of retinoblastoma gene product phosphorylation and
p34cdc2 kinase activation in serum-stimulated human fibroblasts.
J Biol Chem 268:138 –145
34. Boynton AL, Whitfield JF, Isaacs RJ, Tremblay RG 1977 Different
extracellular calcium requirements for proliferation of nonneoplas-
tic, preneoplastic, and neoplastic mouse cells. Cancer Res 37:2657–
2661
35. Swierenga SH, Whitfield JF, Karasaki S 1978 Loss of proliferative
calcium dependence: simple in vitro indicator of tumorigenicity.
Proc Natl Acad Sci USA 75:6069 – 6072
36. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL 1993
Intracellular Ca2⫹ pool content is linked to control of cell growth.
Proc Natl Acad Sci USA 90:4986 – 4990
37. Greber UF, Gerace L 1995 Depletion of calcium from the lumen of
endoplasmic reticulum reversibly inhibits passive diffusion and
signal-mediated transport into the nucleus. J Cell Biol 128:5–14
38. Hussain A, Garnett C, Klein MG, Tsai-Wu JJ, Schneider MF, Inesi
G 1995 Direct involvement of intracellular Ca2⫹ transport ATPase
in the development of thapsigargin resistance by Chinese hamster
lung fibroblasts. J Biol Chem 270:12140 –12146
39. Bootman MD, Collins TJ, Peppiatt CM, Prothero LS, Mackenzie
L, De Smet P, Travers M, Tovey SC, Seo JT, Berridge MJ, Ciccolini
F, Lipp P 2001 Calcium signalling–an overview. Semin Cell Dev
Biol 12:3–10
40. Santella L 1998 The role of calcium in the cell cycle: facts and
hypotheses. Biochem Biophys Res Commun 244:317–324
Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212
by guest
on 28 April 2018
Endocrine Reviews, December 2003, 24(6):719 –736 733
41. Berridge MJ 1995 Calcium signalling and cell proliferation. Bioes-
says 17:491–500
42. Hill TD, Kindmark H, Boynton AL 1988 Epidermal growth factor-
stimulated DNA synthesis requires an influx of extracellular cal-
cium. J Cell Biochem 38:137–144
43. Whitfield JF, Bird RP, Chakravarthy BR, Isaacs RJ, Morley P 1995
Calcium-cell cycle regulator, differentiator, killer, chemopreventor,
and maybe, tumor promoter. J Cell Biochem Suppl 22:74 –91
44. Whitaker M, Larman MG 2001 Calcium and mitosis. Semin Cell
Dev Biol 12:53–58
45. Poenie M, Alderton J, Tsien RY, Steinhardt RA 1985 Changes of
free calcium levels with stages of the cell division cycle. Nature
315:147–149
46. Poenie M, Alderton J, Steinhardt R, Tsien R 1986 Calcium rises
abruptly and briefly throughout the cell at the onset of anaphase.
Science 233:886 – 889
47. Chin D, Means AR 2000 Calmodulin: a prototypical calcium sen-
sor. Trends Cell Biol 10:322–328
48. Tjandra N, Bax A, Crivici A, Ikura M 1999 Calmodulin structure
and target interaction. In: Carafoli E, Klee CB, eds. Calcium as a
cellular regulator. New York: Oxford University Press; 152–169
49. Meador WE, Means AR, Quiocho FA 1992 Target enzyme recog-
nition by calmodulin: 2.4 A structure of a calmodulin-peptide com-
plex. Science 257:1251–1255
50. Meador WE, Means AR, Quiocho FA 1993 Modulation of calmod-
ulin plasticity in molecular recognition on the basis of x-ray struc-
tures. Science 262:1718 –1721
51. Roth SM, Schneider DM, Strobel LA, VanBerkum MF, Means
AR, Wand AJ 1991 Structure of the smooth muscle myosin light-
chain kinase calmodulin-binding domain peptide bound to cal-
modulin. Biochemistry 30:10078 –10084
52. Roth SM, Schneider DM, Strobel LA, Van Berkum MF, Means
AR, Wand AJ 1992 Characterization of the secondary structure of
calmodulin in complex with a calmodulin-binding domain pep-
tide. Biochemistry 31:1443–1451
53. Gnegy ME 1993 Calmodulin in neurotransmitter and hormone
action. Annu Rev Pharmacol Toxicol 33:45–70
54. Chafouleas JG, Bolton WE, Hidaka H, Boyd III AE, Means AR
1982 Calmodulin and the cell cycle: involvement in regulation of
cell-cycle progression. Cell 28:41–50
55. Chafouleas JG, Lagace L, Bolton WE, Boyd III AE, Means AR 1984
Changes in calmodulin and its mRNA accompany reentry of qui-
escent (G0) cells into the cell cycle. Cell 36:73– 81
56. Brooks-Frederich KM, Cianciarulo FL, Rittling SR, Cristofalo VJ
1993 Cell cycle-dependent regulation of Ca2⫹ in young and senes-
cent WI-38 cells. Exp Cell Res 205:412– 415
57. Piol MR, Berchtold MW, Bachs O, Heizmann CW 1988 Increased
calmodulin synthesis in the pre-replicative phase of rat liver re-
generation. FEBS Lett 231:445– 450
58. Agell N, Pujol MJ, Lopez-Girona A, Bosch M, Rosa JL, Bachs O
1994 Calmodulin expression during rat liver regeneration. Hepa-
tology 20:1002–1008
59. Rasmussen CD, Means RL, Lu KP, May GS, Means AR 1990
Characterization and expression of the unique calmodulin gene of
Aspergillus nidulans. J Biol Chem 265:13767–13775
60. Rasmussen CD, Means AR 1987 Calmodulin is involved in reg-
ulation of cell proliferation. EMBO J 6:3961–3968
61. Lu KP, Rasmussen CD, May GS, Means AR 1992 Cooperative
regulation of cell proliferation by calcium and calmodulin in As-
pergillus nidulans. Mol Endocrinol 6:365–374
62. Rasmussen CD, Means AR 1989 Calmodulin is required for cell-
cycle progression during G1 and mitosis. EMBO J 8:73– 82
63. Schmalzigaug R, Ye Q, Berchtold MW 2001 Calmodulin protects
cells from death under normal growth conditions and mitogenic
starvation but plays a mediating role in cell death upon B-cell
receptor stimulation. Immunology 103:332–342
64. Chafouleas JG, Pardue RL, Brinkley BR, Dedman JR, Means AR
1981 Regulation of intracellular levels of calmodulin and tubulin in
normal and transformed cells. Proc Natl Acad Sci USA 78:996 –1000
65. Zendegui JG, Zielinski RE, Watterson DM, Van Eldik LJ 1984
Biosynthesis of calmodulin in normal and virus-transformed
chicken embryo fibroblasts. Mol Cell Biol 4:883– 889
66. Durkin JP, Whitfield JF, MacManus JP 1983 The role of calmod-
 734 Endocrine Reviews, December 2003, 24(6):719 –736
ulin in the proliferation of transformed and phenotypically normal
tsASV-infected rat cells. J Cell Physiol 115:313–319
67. Ye Q, Wei Y, Fischer R, Borner C, Berchtold MW 1997 Expression
of calmodulin and calmodulin binding proteins in rat fibroblasts
stably transfected with protein kinase C and oncogenes. Biochim
Biophys Acta 1359:89 –96
68. Klug M, Blum JK, Ye Q, Berchtold MW 1994 Intracellular Ca2⫹
and Ca2⫹-binding proteins in chemically transformed rat fibro-
blasts. Exp Cell Res 213:313–318
69. Heiman RG, Atkinson RC, Andruss BF, Bolduc C, Kovalick GE,
Beckingham K 1996 Spontaneous avoidance behavior in Drosophila
null for calmodulin expression. Proc Natl Acad Sci USA 93:2420 –
2425
70. Davis TN, Urdea MS, Masiarz FR, Thorner J 1986 Isolation of the
yeast calmodulin gene: calmodulin is an essential protein. Cell
47:423– 431
71. Takeda T, Yamamoto M 1987 Analysis and in vivo disruption of the
gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl
Acad Sci USA 84:3580 –3584
72. Moser MJ, Flory MR, Davis TN 1997 Calmodulin localizes to the
spindle pole body of Schizosaccharomyces pombe and performs an
essential function in chromosome segregation. J Cell Sci 110:1805–
1812
73. Brockerhoff SE, Davis TN 1992 Calmodulin concentrates at re-
gions of cell growth in Saccharomyces cerevisiae. J Cell Biol 118:
619 – 629
74. Ohya Y, Anraku Y 1992 Yeast calmodulin: structural and func-
tional elements essential for the cell cycle. Cell Calcium 13:445– 455
75. Ohya Y, Anraku Y 1989 A galactose-dependent cmd1 mutant of
Saccharomyces cerevisiae: involvement of calmodulin in nuclear di-
vision. Curr Genet 15:113–120
76. Davis TN 1992 A temperature-sensitive calmodulin mutant loses
viability during mitosis. J Cell Biol 118:607– 617
77. Ohya Y, Botstein D 1994 Diverse essential functions revealed by
complementing yeast calmodulin mutants. Science 263:963–966
78. Lu KP, Osmani SA, Osmani AH, Means AR 1993 Essential roles
for calcium and calmodulin in G2/M progression in Aspergillus
nidulans. J Cell Biol 121:621– 630
79. Toutenhoofd SL, Strehler EE 2000 The calmodulin multigene fam-
ily as a unique case of genetic redundancy: multiple levels of
regulation to provide spatial and temporal control of calmodulin
pools? Cell Calcium 28:83–96
80. Nojima H 1989 Structural organization of multiple rat calmodulin
genes. J Mol Biol 208:269 –282
81. Nojima H, Sokabe H 1989 Structural organization of calmodulin
genes in the rat genome. Adv Exp Med Biol 255:223–232
82. Berchtold MW, Egli R, Rhyner JA, Hameister H, Strehler EE 1993
Localization of the human bona fide calmodulin genes CALM1,
CALM2, and CALM3 to chromosomes 14q24 – q31,2p21.1-p21.3,
and 19q13.2-q13.3. Genomics 16:461– 465
83. Reddy GP, Reed WC, Sheehan E, Sacks DB 1992 Calmodulin-
specific monoclonal antibodies inhibit DNA replication in mam-
malian cells. Biochemistry 31:10426 –10430
84. Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, Nagata
T 1981 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a
calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad
Sci USA 78:4354 – 4357
85. Taules M, Rius E, Talaya D, Lopez-Girona A, Bachs O, Agell N
1998 Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4)
activity and nuclear accumulation of cyclin D1-Cdk4 during G1.
J Biol Chem 273:33279 –33286
86. Lopez-Girona A, Colomer J, Pujol MJ, Bachs O, Agell N 1992
Calmodulin regulates DNA polymerase ␣ activity during prolif-
erative activation of NRK cells. Biochem Biophys Res Commun
184:1517–1523
87. Wei JW, Hickie RA, Klaassen DJ 1983 Inhibition of human breast
cancer colony formation by anticalmodulin agents: trifluoperazine,
W-7, and W-13. Cancer Chemother Pharmacol 11:86 –90
88. Taules M, Rodriguez-Vilarrupla A, Rius E, Estanyol JM, Casano-
vas O, Sacks DB, Perez-Paya E, Bachs O, Agell N 1999 Calmodulin
binds to p21(Cip1) and is involved in the regulation of its nuclear
localization. J Biol Chem 274:24445–24458
89. Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Rous-
Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212
by guest
on 28 April 2018
Kahl and Means • Calcium/Calmodulin and the Cell Cycle
sel MF, Sherr CJ 1993 Overexpression of mouse D-type cyclins
accelerates G1 phase in rodent fibroblasts. Genes Dev 7:1559 –1571
90. Musgrove EA, Lee CS, Buckley MF, Sutherland RL 1994 Cyclin
D1 induction in breast cancer cells shortens G1 and is sufficient for
cells arrested in G1 to complete the cell cycle. Proc Natl Acad Sci
USA 91:8022– 8026
91. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G 1993 Cyclin
D1 is a nuclear protein required for cell cycle progression in G1.
Genes Dev 7:812– 821
92. Li F, Wang X, Capasso JM, Gerdes AM 1996 Rapid transition of
cardiac myocytes from hyperplasia to hypertrophy during post-
natal development. J Mol Cell Cardiol 28:1737–1746
93. Swierenga SH, MacManus JP, Whitfield JF 1976 Regulation by
calcium of the proliferation of heart cells from young adult rats. In
Vitro 12:31–36
94. Gruver CL, DeMayo F, Goldstein MA, Means AR 1993 Targeted
developmental overexpression of calmodulin induces proliferative
and hypertrophic growth of cardiomyocytes in transgenic mice.
Endocrinology 133:376 –388
95. Cyert MS 2001 Genetic analysis of calmodulin and its targets in
Saccharomyces cerevisiae. Annu Rev Genet 35:647– 672
96. Luan Y, Matsuura I, Yazawa M, Nakamura T, Yagi K 1987 Yeast
calmodulin: structural and functional differences compared with
vertebrate calmodulin. J Biochem (Tokyo) 102:1531–1537
97. Geiser JR, van Tuinen D, Brockerhoff SE, Neff MM, Davis TN
1991 Can calmodulin function without binding calcium? Cell 65:
949 –959
98. Geiser JR, Sundberg HA, Chang BH, Muller EG, Davis TN 1993
The essential mitotic target of calmodulin is the 110-kilodalton
component of the spindle pole body in Saccharomyces cerevisiae. Mol
Cell Biol 13:7913–7924
99. Stirling DA, Stark MJ 2000 Mutations in SPC110, encoding the
yeast spindle pole body calmodulin-binding protein, cause defects
in cell integrity as well as spindle formation. Biochim Biophys Acta
1499:85–100
100. Stirling DA, Welch KA, Stark MJ 1994 Interaction with calmod-
ulin is required for the function of Spc110p, an essential component
of the yeast spindle pole body. EMBO J 13:4329 – 4342
101. Moser MJ, Lee SY, Klevit RE, Davis TN 1995 Ca2⫹ binding to
calmodulin and its role in Schizosaccharomyces pombe as revealed by
mutagenesis and NMR spectroscopy. J Biol Chem 270:20643–20652
102. Flory MR, Morphew M, Joseph JD, Means AR, Davis TN 2002
Pcp1p, an Spc110p-related calmodulin target at the centrosome of
the fission yeast Schizosaccharomyces pombe. Cell Growth Differ 13:
47–58
103. McGoldrick CA, Gruver C, May GS 1995 myoA Of Aspergillus
nidulans encodes an essential myosin I required for secretion and
polarized growth. J Cell Biol 128:577–587
104. Joseph JD, Means AR 2000 Identification and characterization of
two Ca2⫹/CaM-dependent protein kinases required for normal
nuclear division in Aspergillus nidulans. J Biol Chem 275:38230 –
38238
105. Dayton JS, Means AR 1996 Ca2⫹/calmodulin-dependent kinase is
essential for both growth and nuclear division in Aspergillus nidu-
lans. Mol Biol Cell 7:1511–1519
106. Rasmussen C, Garen C, Brining S, Kincaid RL, Means RL, Means
AR 1994 The calmodulin-dependent protein phosphatase catalytic
subunit (calcineurin A) is an essential gene in Aspergillus nidulans.
EMBO J 13:2545–2552
107. Guerini D 1997 Calcineurin: not just a simple protein phosphatase.
Biochem Biophys Res Commun 235:271–275
108. Klee CB, Wang X, Ren H 1999 Calcium-regulated protein dephos-
phorylation. In: Carafoli E, Klee CB, eds. Calcium as a cellular
regulator. New York: Oxford University Press; 344 –363
109. Klee CB, Ren H, Wang X 1998 Regulation of the calmodulin-
stimulated protein phosphatase, calcineurin. J Biol Chem
273:13367–13370
110. Aramburu J, Rao A, Klee CB 2000 Calcineurin: from structure to
function. Curr Top Cell Regul 36:237–295
111. Cardenas ME, Heitman J 1995 Role of calcium in T-lymphocyte
activation. Adv Second Messenger Phosphoprotein Res 30:281–298
112. Ho S, Clipstone N, Timmermann L, Northrop J, Graef I,
Fiorentino D, Nourse J, Crabtree GR 1996 The mechanism of
 Kahl and Means • Calcium/Calmodulin and the Cell Cycle
action of cyclosporin A and FK506. Clin Immunol Immunopathol
80:S40 –S45
113. Schreiber SL, Crabtree GR 1992 The mechanism of action of cy-
closporin A and FK506. Immunol Today 13:136 –142
114. Boustany LM, Cyert MS 2002 Calcineurin-dependent regulation of
Crz1p nuclear export requires Msn5p and a conserved calcineurin
docking site. Genes Dev 16:608 – 619
115. Stathopoulos-Gerontides A, Guo JJ, Cyert MS 1999 Yeast cal-
cineurin regulates nuclear localization of the Crz1p transcription
factor through dephosphorylation. Genes Dev 13:798 – 803
116. Yoshimoto H, Saltsman K, Gasch AP, Li HX, Ogawa N, Botstein
D, Brown PO, Cyert MS 2002 Genome-wide analysis of gene
expression regulated by the calcineurin/Crz1p signaling pathway
in Saccharomyces cerevisiae. J Biol Chem 277:31079 –31088
117. Withee JL, Mulholland J, Jeng R, Cyert MS 1997 An essential role
of the yeast pheromone-induced Ca2⫹ signal is to activate cal-
cineurin. Mol Biol Cell 8:263–277
118. Sugiura R, Sio SO, Shuntoh H, Kuno T 2002 Calcineurin phos-
phatase in signal transduction: lessons from fission yeast. Genes
Cells 7:619 – 627
119. Yoshida T, Toda T, Yanagida M 1994 A calcineurin-like gene
ppb1⫹ in fission yeast: mutant defects in cytokinesis, cell polarity,
mating and spindle pole body positioning. J Cell Sci 107:1725–1735
120. Plochocka-Zulinska D, Rasmussen G, Rasmussen C 1995 Regu-
lation of calcineurin gene expression in Schizosaccharomyces pombe.
Dependence on the ste11 transcription factor. J Biol Chem 270:
24794 –24799
121. Fox DS, Cruz MC, Sia RA, Ke H, Cox GM, Cardenas ME, Heitman
J 2001 Calcineurin regulatory subunit is essential for virulence and
mediates interactions with FKBP12-FK506 in Cryptococcus neofor-
mans. Mol Microbiol 39:835– 849
122. Odom A, Muir S, Lim E, Toffaletti DL, Perfect J, Heitman J 1997
Calcineurin is required for virulence of Cryptococcus neoformans.
EMBO J 16:2576 –2589
123. Fox DS, Heitman J 2002 Good fungi gone bad: the corruption of
calcineurin. Bioessays 24:894 –903
124. Cruz MC, Fox DS, Heitman J 2001 Calcineurin is required for
hyphal elongation during mating and haploid fruiting in Crypto-
coccus neoformans. EMBO J 20:1020 –1032
125. Nanthakumar NN, Dayton JS, Means AR 1996 Role of Ca⫹⫹/
calmodulin binding proteins in Aspergillus nidulans cell cycle reg-
ulation. Prog Cell Cycle Res 2:217–228
126. Clipstone NA, Crabtree GR 1992 Identification of calcineurin as a
key signalling enzyme in T-lymphocyte activation. Nature 357:
695– 697
127. Fruman DA, Klee CB, Bierer BE, Burakoff SJ 1992 Calcineurin
phosphatase activity in T lymphocytes is inhibited by FK 506 and
cyclosporin A. Proc Natl Acad Sci USA 89:3686 –3690
128. Liu J, Farmer Jr JD, Lane WS, Friedman J, Weissman I, Schreiber
SL 1991 Calcineurin is a common target of cyclophilin-cyclosporin
A and FKBP-FK506 complexes. Cell 66:807– 815
129. Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ,
Cohen P, Mackintosh C, Klee CB, Schreiber SL 1992 Inhibition of
T cell signaling by immunophilin-ligand complexes correlates with
loss of calcineurin phosphatase activity. Biochemistry 31:3896 –
3901
130. Furue M, Gaspari AA, Katz SI 1988 The effect of cyclosporin A on
epidermal cells. II. Cyclosporin A inhibits proliferation of normal
and transformed keratinocytes. J Invest Dermatol 90:796 – 800
131. Sharpe GR, Fisher C 1990 Time-dependent inhibition of growth of
human keratinocytes and fibroblasts by cyclosporin A: effect on
keratinocytes at therapeutic blood levels. Br J Dermatol 123:207–213
132. Thyberg J, Hansson GK 1991 Cyclosporine A inhibits induction of
DNA synthesis by PDGF and other peptide mitogens in cultured
rat aortic smooth muscle cells and dermal fibroblasts. Growth
Factors 4:209 –219
133. Richter A, Davies DE, Alexander P 1995 Growth inhibitory effects
of FK506 and cyclosporin A independent of inhibition of cal-
cineurin. Biochem Pharmacol 49:367–373
134. Tomono M, Toyoshima K, Ito M, Amano H 1996 Calcineurin is
essential for DNA synthesis in Swiss 3T3 fibroblasts. Biochem J
317:675– 680
135. Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lag-
Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212
by guest
on 28 April 2018
Endocrine Reviews, December 2003, 24(6):719 –736 735
man M, Shimbo T, Suthanthiran M 1999 Cyclosporine induces
cancer progression by a cell-autonomous mechanism. Nature 397:
530 –534
136. Khanna AK, Hosenpud JD 1999 Cyclosporine induces the expres-
sion of the cyclin inhibitor p21. Transplantation 67:1262–1268
137. Tomono M, Toyoshima K, Ito M, Amano H, Kiss Z 1998 Inhibitors
of calcineurin block expression of cyclins A and E induced by
fibroblast growth factor in Swiss 3T3 fibroblasts. Arch Biochem
Biophys 353:374 –378
138. Schneider G, Oswald F, Wahl C, Greten FR, Adler G, Schmid RM
2002 Cyclosporine inhibits growth through the activating tran-
scription factor/cAMP-responsive element-binding protein bind-
ing site in the cyclin D1 promoter. J Biol Chem 277:43599 – 43607
139. Baksh S, Widlund HR, Frazer-Abel AA, Du J, Fosmire S, Fisher
DE, DeCaprio JA, Modiano JF, Burakoff SJ 2002 NFATc2-medi-
ated repression of cyclin-dependent kinase 4 expression. Mol Cell
10:1071–1081
140. Baksh S, DeCaprio JA, Burakoff SJ 2000 Calcineurin regulation of
the mammalian G0/G1 checkpoint element, cyclin dependent ki-
nase 4. Oncogene 19:2820 –2827
141. Crabtree GR 1999 Generic signals and specific outcomes: signaling
through Ca2⫹, calcineurin, and NF-AT. Cell 96:611– 614
142. Vogel KW, Briesewitz R, Wandless TJ, Crabtree GR 2001 Cal-
cineurin inhibitors and the generalization of the presenting protein
strategy. Adv Protein Chem 56:253–291
143. Bram RJ, Hung DT, Martin PK, Schreiber SL, Crabtree GR 1993
Identification of the immunophilins capable of mediating inhibi-
tion of signal transduction by cyclosporin A and FK506: roles of
calcineurin binding and cellular location. Mol Cell Biol 13:4760 –
4769
144. Kung L, Halloran PF 2000 Immunophilins may limit calcineurin
inhibition by cyclosporine and tacrolimus at high drug concentra-
tions. Transplantation 70:327–335
145. Schulman H, Braun A 1999 Calcium/calmodulin-dependent pro-
tein kinases. In: Carafoli E, Klee CB, eds. Calcium as a cellular
regulator. New York: Oxford University Press; 311–335
146. Means AR 2000 Regulatory cascades involving calmodulin-depen-
dent protein kinases. Mol Endocrinol 14:4 –13
147. Hook SS, Means AR 2001 Ca2⫹/CaM-dependent kinases: from
activation to function. Annu Rev Pharmacol Toxicol 41:471–505
148. Soderling TR, Stull JT 2001 Structure and regulation of calcium/
calmodulin-dependent protein kinases. Chem Rev 101:2341–2352
149. Soderling TR, Chang B, Brickey D 2001 Cellular signaling through
multifunctional Ca2⫹/calmodulin-dependent protein kinase II.
J Biol Chem 276:3719 –3722
150. Hudmon A, Schulman H 2002 Structure-function of the multi-
functional Ca2⫹/calmodulin-dependent protein kinase II. Biochem
J 364:593– 611
151. Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H 2001
Interaction with the NMDA receptor locks CaMKII in an active
conformation. Nature 411:801– 805
152. Soderling TR 1999 The Ca-calmodulin-dependent protein kinase
cascade. Trends Biochem Sci 24:232–236
153. Hook SS, Kemp BE, Means AR 1999 Peptide specificity determi-
nants at P-7 and P-6 enhance the catalytic efficiency of Ca2⫹/
calmodulin-dependent protein kinase I in the absence of activation
loop phosphorylation. J Biol Chem 274:20215–20222
154. Tombes RM, Mikkelsen RB, Jarvis WD, Grant S 1999 Down-
regulation of ␦ CaM kinase II in human tumor cells. Biochim Bio-
phys Acta 1452:1–11
155. Tombes RM, Krystal GW 1997 Identification of novel human tu-
mor cell-specific CaMK-II variants. Biochim Biophys Acta 1355:
281–292
156. Shang S, Takai N, Nishida M, Miyazaki T, Nasu K, Miyakawa I
2003 CaMKIV expression is associated with clinical stage and
PCNA-labeling index in endometrial carcinoma. Int J Mol Med
11:181–186
157. Takai N, Miyazaki T, Nishida M, Nasu K, Miyakawa I 2002
Ca2⫹/calmodulin-dependent protein kinase IV expression in epi-
thelial ovarian cancer. Cancer Lett 183:185–193
158. Williams CL, Phelps SH, Porter RA 1996 Expression of Ca2⫹/
calmodulin-dependent protein kinase types II and IV, and reduced
DNA synthesis due to the Ca2⫹/calmodulin-dependent protein
 736 Endocrine Reviews, December 2003, 24(6):719 –736
kinase inhibitor KN-62 (1-[N, O-bis(5-isoquinolinesulfonyl)-N-
methyl-l-tyrosyl]-4-phenyl piperazine) in small cell lung carci-
noma. Biochem Pharmacol 51:707–715
159. Corcoran EE, Joseph JD, MacDonald JA, Kane CD, Haystead TA,
Means AR 2003 Proteomic analysis of calcium/calmodulin-depen-
dent protein kinase I and IV in vitro substrates reveals distinct
catalytic preferences. J Biol Chem 278:10516 –10522
160. Anderson KA, Means RL, Huang QH, Kemp BE, Goldstein EG,
Selbert MA, Edelman AM, Fremeau RT, Means AR 1998 Com-
ponents of a calmodulin-dependent protein kinase cascade. Mo-
lecular cloning, functional characterization and cellular localization
of Ca2⫹/calmodulin-dependent protein kinase kinase ␤. J Biol
Chem 273:31880 –31889
161. Tokumitsu H, Iwabu M, Ishikawa Y, Kobayashi R 2001 Differ-
ential regulatory mechanism of Ca2⫹/calmodulin-dependent pro-
tein kinase kinase isoforms. Biochemistry 40:13925–13932
162. Tokumitsu H, Muramatsu M, Ikura M, Kobayashi R 2000 Reg-
ulatory mechanism of Ca2⫹/calmodulin-dependent protein kinase
kinase. J Biol Chem 275:20090 –20095
163. Pausch MH, Kaim D, Kunisawa R, Admon A, Thorner J 1991
Multiple Ca2⫹/calmodulin-dependent protein kinase genes in a
unicellular eukaryote. EMBO J 10:1511–1522
164. Ohya Y, Kawasaki H, Suzuki K, Londesborough J, Anraku Y 1991
Two yeast genes encoding calmodulin-dependent protein kinases.
Isolation, sequencing and bacterial expressions of CMK1 and
CMK2. J Biol Chem 266:12784 –12794
165. Miyakawa T, Oka Y, Tsuchiya E, Fukui S 1989 Saccharomyces
cerevisiae protein kinase dependent on Ca2⫹ and calmodulin. J
Bacteriol 171:1417–1422
166. Moser MJ, Geiser JR, Davis TN 1996 Ca2⫹-calmodulin promotes
survival of pheromone-induced growth arrest by activation of cal-
cineurin and Ca2⫹-calmodulin-dependent protein kinase. Mol Cell
Biol 16:4824 – 4831
167. Rasmussen CD 2000 Cloning of a calmodulin kinase I homologue
from Schizosaccharomyces pombe. J Biol Chem 275:685– 690
168. Alemany V, Sanchez-Piris M, Bachs O, Aligue R 2002 Cmk2, a
novel serine/threonine kinase in fission yeast. FEBS Lett 524:79 – 86
169. Bartelt DC, Fidel S, Farber LH, Wolff DJ, Hammell RL 1988
Calmodulin-dependent multifunctional protein kinase in Aspergil-
lus nidulans. Proc Natl Acad Sci USA 85:3279 –3283
170. Rasmussen G, Rasmussen C 1995 Calmodulin-dependent protein
kinase II is required for G1/S progression in HeLa cells. Biochem
Cell Biol 73:201–207
171. Tombes RM, Grant S, Westin EH, Krystal G 1995 G1 cell cycle
arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an
inhibitor of CaMK-II (the multifunctional Ca2⫹/CaM kinase). Cell
Growth Differ 6:1063–1070
172. Morris TA, DeLorenzo RJ, Tombes RM 1998 CaMK-II inhibition
reduces cyclin D1 levels and enhances the association of p27 kip1
with Cdk2 to cause G1 arrest in NIH 3T3 cells. Exp Cell Res 240:
218 –227
173. Ohta Y, Ohba T, Miyamoto E 1990 Ca2⫹/calmodulin-dependent
Downloaded from https://academic.oup.com/edrv/article-abstract/24/6/719/2567212
by guest
on 28 April 2018
Kahl and Means • Calcium/Calmodulin and the Cell Cycle
protein kinase II: localization in the interphase nucleus and the
mitotic apparatus of mammalian cells. Proc Natl Acad Sci USA
87:5341–5345
174. Matsumoto Y, Maller JL 2002 Calcium, calmodulin, and CaMKII
requirement for initiation of centrosome duplication in Xenopus egg
extracts. Science 295:499 –502
175. Patel R, Holt M, Philipova R, Moss S, Schulman H, Hidaka H,
Whitaker M 1999 Calcium/calmodulin-dependent phosphoryla-
tion and activation of human Cdc25-C at the G2/M phase transition
in HeLa cells. J Biol Chem 274:7958 –7968
176. Jackman MR, Pines JN 1997 Cyclins and the G2/M transition.
Cancer Surv 29:47–73
177. Nilsson I, Hoffmann I 2000 Cell cycle regulation by the Cdc25
phosphatase family. Prog Cell Cycle Res 4:107–114
178. Petzelt CP, Kodirov S, Taschenberger G, Kox WJ 2001 Participa-
tion of the Ca2⫹-calmodulin-activated kinase II in the control of
metaphase-anaphase transition in human cells. Cell Biol Int 25:
403– 409
179. Dayton JS, Sumi M, Nanthakumar NN, Means AR 1997 Expres-
sion of a constitutively active Ca2⫹/calmodulin-dependent kinase
in Aspergillus nidulans spores prevents germination and entry into
the cell cycle. J Biol Chem 272:3223–3230
180. Lorca T, Galas S, Fesquet D, Devault A, Cavadore JC, Doree M
1991 Degradation of the proto-oncogene product p39 mos is not
necessary for cyclin proteolysis and exit from meiotic metaphase:
requirement for a Ca2⫹-calmodulin dependent event. EMBO J 10:
2087–2093
181. Lorca T, Abrieu A, Means A, Doree M 1994 Ca2⫹ is involved
through type II calmodulin-dependent protein kinase in cyclin
degradation and exit from metaphase. Biochim Biophys Acta 1223:
325–332
182. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means
A, Doree M 1993 Calmodulin-dependent protein kinase II mediates
inactivation of MPF and CSF upon fertilization of Xenopus eggs.
Nature 366:270 –273
183. Sanchez-Piris M, Posas F, Alemany V, Winge I, Hidalgo E, Bachs
O, Aligue R 2002 The serine/threonine kinase Cmk2 is required for
oxidative stress response in fission yeast. J Biol Chem 277:17722–
17727
184. Hidaka H, Yokokura H 1996 Molecular and cellular pharmacology
of a calcium/calmodulin-dependent protein kinase II (CaM kinase
II) inhibitor, KN-62, and proposal of CaM kinase phosphorylation
cascades. Adv Pharmacol 36:193–219
185. White RR, Kwon YG, Taing M, Lawrence DS, Edelman AM 1998
Definition of optimal substrate recognition motifs of Ca2⫹-calmod-
ulin-dependent protein kinases IV and II reveals shared and dis-
tinctive features. J Biol Chem 273:3166 –3172
186. Dale S, Wilson WA, Edelman AM, Hardie DG 1995 Similar sub-
strate recognition motifs for mammalian AMP-activated protein
kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1,
and mammalian calmodulin-dependent protein kinase I. FEBS Lett
361:191–195