Protein Prenylation

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POST-TRANSLATIONAL MODIFICATIONS
Protein prenylation: unique fats make

their mark on biology
Mei Wang1 and Patrick J.Casey1,2
Abstract | The modification of eukaryotic proteins by isoprenoid lipids, which is known as

prenylation, controls the localization and activity of a range of proteins that have crucial
functions in biological regulation. The roles of prenylated proteins in cells are well conserved
across species, underscoring the biological and evolutionary importance of this lipid modification
pathway. Genetic suppression and pharmacological inhibition of the protein prenylation
machinery have provided insights into several cellular processes and into the aetiology of
diseases in which prenylation is involved. The functional dependence of prenylation substrates,
such as RAS proteins, on this modification and the therapeutic potential of targeting the
prenylation process in pathological conditions accentuate the need to fully understand this form
of post-translational modification.
CAAX proteins
Proteins containing a CAAX
motif at their carboxyl termini,
in which C is the Cys residue
that functions as the
isoprenoid attachment site,
Asignifies any aliphatic amino
acid, and X denotes any of
several amino acids.
Program in Cancer and Stem

Cell Biology, Duke-NUS
Medical School,
Singapore169857.
2
Department of
Pharmacology and Cancer
Biology, Duke University
Medical Center, Durham,
North Carolina 27710, USA.
mei.wang@duke-nus.edu.sg;
patrick.casey@duke.edu
1
doi:10.1038/nrm.2015.11
Published online 21 Jan 2016
Life would have been so much simpler for molecular and

cell biologists if proteins were straightforward translations of the genetic code using the 20 or more l-amino
acids that living organisms produce and use. However,
this simple translation from nucleic acid sequence to
polypeptide sequence was apparently insufficient to support evolution beyond the most primitive of stages. Over
the past billion years, organisms have developed a vast
array of co-translational and post-translational modifications to expand and enhance the sophistication of protein functions in response to complex external stimuli
and internal adjustments1.
One example of such a post-translational modification is prenylation (that is, modification by isoprenoid
lipids). This processing pathway makes a crucial contribution to the functions of a select group of proteins
that are involved in biological regulation in eukaryotic
cells2,3. Most prenylated proteins are CAAX proteins,
for which prenylation is initiated by the attachment
of a 15carbon (farnesyl) or a 20carbon (geranylgeranyl) isoprenoid lipid to the Cys residue by protein
farnesyltransferase (FTase) or protein geranlygeranyltransferaseI (GGTase I), respectively. The enzymatic
reaction is termed farnesylation if it involves the farnesyl
isoprenoid, or geranylgeranylation if it involves the
geranylgeranyl isoprenoid. Proteins can then be further
processed by RAS-converting CAAX endopeptidase 1
(RCE1), which removes the -AAX residues, followed by
isoprenylcysteine carboxylmethyltransferase (ICMT),
which caps the carboxyl group on the now carboxy
terminal isoprenoid-modified Cys residue with a methyl
group4,5(FIG.1).
Prenylation provides proteins with a hydrophobic

C terminus, the consequence of which is a greatly
increased capacity to interact with cellular membranes,
which have a high concentration of signalling molecules.
Together, the prenylated motif and the individual CAAX
protein sequence and structure determine the discrete
localization of the protein either to a specific area of
the plasma membrane or to the endomembrane, or even
to a cytosolic location. Modification of the C termini of
these proteins affects many of their functions, in addition to localizing the proteins to different compartments
of the cell, including facilitating specific proteinprotein
interactions and modulating protein stability. The regulatory capacity of prenylated proteins in cell signalling 6,
and the mounting evidence of their essential roles in
various disease processes7, have driven efforts to elucidate the biochemical processes involved in prenylation
and its effects on the biological functions of the proteins.
In this Review, we dissect the three steps prenyl
ation, proteolysis and methylation that are involved in
this post-translational modification, from biochemistry
to functional effects on the target CAAX proteins, and
discuss the relevance of this processing to human diseases. Although many of the early discoveries on prenyl
ation were in simpler eukaryotic organisms8, the focus
of this Review is the mammalian enzymes and their
effects on mammalian cell functions, because much
of the recent understanding of protein prenylation has
been driven by the study of pathophysiologies. One
example is the rare disease progeria, the pathology of
which is a direct consequence of the toxic accumulation
of prenylated prelamin A protein. The recognition that
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a
Ribosome
PP
Cholesterol
GGPP
SH
CAAX
GGPP
synthase
Squalene
RAS
Or
PP
Protein
prenyltransferases
FPP
FPP
synthase
PPi
GPP
Tracking
S
CAAX
RAS
GPP
synthase
AdoMet AdoHcy
RAS CCOO
S
AAX
IPP
RAS CCOO CH3

S
RCE1
ER membrane
Mevalonate
HMG-CoA
reductase
Acetyl-CoA
ICMT
Statins
HMG-CoA
Figure 1 | Isoprenoid metabolism and CAAX protein prenylation. a | The production

farnesyl| Molecular
diphosphate
(FPP)
and
Natureof
Reviews
Cell
Biology
geranylgeranyl diphosphate (GGPP) share the early steps of the cholesterol synthesis pathway initiated by HMG-CoA
reductase, which is an endoplasmic reticulum (ER)associated enzyme and the target of statin drugs. FPP can be
converted either to squalene (which is the committed precursor of sterols) or to GGPP, by squalene synthase or GGPP
synthase, respectively. b | Cytosolic protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase),
which are the CAAX protein prenyltransferases, add farnesyl or geranylgeranyl, respectively, to the Cys of the CAAX motif
of substrate proteins such as RAS. The post-prenylation modifications occur on the cytosolic surface of the ER as a result
of the actions of RAS-converting CAAX endopeptidase 1 (RCE1) and isoprenylcysteine carboxylmethyltransferase (ICMT),
both of which are intrinsic ER membrane proteins. The fully processed prenylated proteins are then trafficked to the
destination membrane. AdoHcy, Sadenosyl-lhomocysteine; AdoMet, Sadenosyl methionine; GPP, geranyl diphosphate;
IPP, isopentenyl diphosphate.
HMG-CoA reductase
(HMGCR). The rate-controlling
enzyme of the mevalonate
pathway of cholesterol and
isoprenoid biosynthesis and
the target of the widely-used
statin drugs.
Sterols
The major biosynthetic end
products of the isoprenoid
biosynthetic pathway; sterols
occur naturally in eukaryotic
organisms, with the most
well-recognized animal sterol
being cholesterol.
Mevalonate
The direct product of the
HMG-CoA reductase (HMGCR)
reaction and the first
committed intermediate in the
biosynthetic pathway that
produces isoprenoids and,
subsequently, sterols.
the maturation of prelamin A depends on prenylation,

combined with understanding of the biochemistry
of prelamin A processing 9,10, led to a swift translation
from bench to bedside. In the cancer field, the prenyl
ation of RAS GTPases, which drive more than 30%
of human cancers, has been the subject of an intense
drug-development effort for more than two decades.
There has been renewed interest of late in targeting
these undruggable RAS proteins, not only because of
advances in the structural determination of RAS and its
partners, but also owing to its importance as a potential
anticancer target. Targeting RAS localization through
the disruption of prenylation remains a viable way to
render this protein druggable (REF.11).
A brief history of protein prenylation

The first evidence for the modification of proteins by
isoprenoid lipids came in the late 1970s from chemical
analysis of a fungal mating factor, rhodotorucine A,
which identified a farnesyl isoprenoid linked to a Cys
residue of the peptide by a thioether bond12. The existence of prenylated proteins in mammals was first suggested by studies of inhibitors and of feedback regulation
of HMG-CoA reductase (HMGCR)13 (FIG.1). The inhibitors,
known as statins, were found to block cell proliferation
in a way that could not be reversed by adding sterols14,15

to the media. This suggested that an early intermediate
(or intermediates) in the biosynthesis of sterols was
involved in the control of cell proliferation. An important clue came from the observation that 3H-labelled
mevalonate was incorporated into many cellular proteins when it was added to the media of statin-treated
cells; these labelled proteins were dubbed prenylated
proteins (REF.16). Subsequently, the nuclear protein
lamin B was the first prenylated protein to be identified
in mammals17, although at that time the identity of the
isoprenoid attached to the protein was notknown.
The important finding that brought prenylation
to theattention of the broader cell biology community wasthe rediscovery in 1988 of the prenylation of
fungal mating peptides specifically, the farnesylation
of the mating pheromone afactor of Saccharomyces
cerevisiae 18. The recognition that the genes encoding
the yeast a factor and lamin B both code for sequences
that predict a CAAX motif at their C termini, and that
disruption of genes in yeast that affected prenylation
processing of the mating factor also affected the function
of another CAAX-type protein, Ras1 (REF.8), prompted
a flurry of activity to determine whether mammalian RAS proteins are also processed in this manner.
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Prenylome
The repertoire of proteins in an
organism that contain
covalently attached
isoprenoidlipids.
Thesubsequent discoveries that human RAS proteins

are indeed farnesylated, and that the modification is
required for oncogenic RAS to transform cells1921, triggered widespread interest in this new form of lipid modification. It was soon shown that either the farnesyl or the
geranylgeranyl isoprenoids could be attached to CAAX
proteins (discussed furtherbelow)22,23.
The three enzymatic steps of protein prenylation

Genomic, structural, biochemical and functional analyses
predict that more than 200 CAAX-type prenylated proteins are expressed by mammalian cells3,2426. In addition,
a mechanistically distinct modification of RAB GTPases
which have crucial roles in vesicular trafficking in
mammalian cells by RAB GGTase II-mediated geranyl
geranylation was also identified27. This Review focuses
on prenylation of the CAAX-type proteins, as the role of
prenylation (and associated methylation) in RAB GTPase
function has been discussed extensively elsewhere28,29.
Comparison of the gene encoding the fungal mating
factor with its product peptide led to the elucidation of
a three-step modification process for CAAX-containing
proteins, as the final form of the peptide lacked
thethree Cterminal residues (AAX) encoded in the
open reading frame of the gene and had a carboxy
methyl m
oiety on the now-Cterminal prenylcysteine.
These two post-prenylation steps proteolysis of the
Cterminal tripeptide and methylation of the prenyl
cysteine were later shown to be catalysed by RCE1 and
ICMT,respectively 5,3032.
Prenylation by FTase and GGTase I. Isoprenoids the
15carbon farnesyl diphosphate (FPP) and the 20carbon
geranylgeranyl diphosphate (GGPP) are built up in
5carbon units from isopentenyl diphosphate33, which
Box 1 | Techniques for the identification and analysis of prenylated proteins
The chemical biologist trying to find lipids attached to proteins has not had a simple task.
The most commonly used approach has involved culturing cells with radioisotope-labelled
precursors of the lipids of interest (3H-mevalonic acid in the case of prenylated proteins)
and then isolating the protein of interest using immunoprecipitation or other bioaffinity
methods, followed by analysis and identification of the attached radioactive lipid157,158. As
well as the use of radioisotopes, a major drawback of this approach is that it examines the
modification on a protein-byprotein basis. In the past few years, several groups have
developed innovative chemical approaches to circumvent these problems and to enable
the detection of prenylated proteins on a larger scale. These methods include the use of
azido- and alkyne-modified farnesyl and geranylgeranyl analogues that can be taken up
by cells and incorporated into cellular proteins; these proteins can then be identified or
captured using agents that are chemoselective for the modification group157,159. In
addition to providing ways to identify new prenylated proteins, these technologies have
increased the likelihood that we may soon be able to directly image prenylated proteins,
and possibly other lipid-modified proteins, in living cells. In a further refinement of this
approach, protein prenyltransferases were engineered to use biotin-modified isoprenoids;
this reaction was coupled with mass spectrometry to examine the mammalian
prenylome160. Another approach to identify novel prenylated proteins is to use synthetic
peptides with different combinations of amino acids in the -AAX sequence to define
possible CAAX combinations that function as efficient substrates for prenyltransferases26.
Expanding the repertoire of protein substrates of the prenylation pathway will increase
our understanding not only of the cellular functions that are affected by this
post-translational modification, but also of the pathophysiological conditions that might
be alleviated by targeting the enzymes ofthis pathway.
is synthesized downstream of mevalonate (FIG.1). Both

FTase and GGTase I are cytosolic enzymes that first
bind to the appropriate isoprenoid through a hydrophobic pocket and then bind to the CAAX sequence of the
protein substrate in an adjacent region3436. The X of
the CAAX motif is the primary determinant of which
of the two enzymes modify the protein, although there
is some overlap and flexibility 2,3,3739. Protein substrates
for FTase in mammalian cells include HRAS, KRAS and
NRAS, prelamin A and lamin B, several proteins involved
in visual signal transduction, and at least three protein
kinases and phosphatases. Known targets of GGTase I
include a large number of RAS-related GTPases, such
as members of the RAS and RHORAC families, and
some other regulatory proteins3,7,37. Given the inherent
difficulties of chemical analysis of the lipid-modification
status of proteins, the primary route for initial prediction of most CAAX motif-containing proteins has been
to deduce this from the DNA coding sequences; however,
emerging chemical techniques are leading to the direct
identification of prenylated proteins in cells and extracts
(BOX1). It remains a challenging task to identify prenyl
ated proteins and to define the functional effect of each of
the three modification steps on these proteins.
Although the two protein prenyltransferases generally
have high specificity for their target CAAX sequences,
there are cases in which they act on each o
thers substrates. An early invivo study showed that overexpression
of GGTase I in a strain of yeast lacking FTase increased
the plasma membrane localization of Ras, which is an
FTase substrate under physiological conditions. Ras is
crucial for yeast survival and proliferation, and modification by the highly expressed GGTaseI rescued growth
defects caused by the elimination of FTase40. In a similar
manner, overexpression of specific CAAX proteins that
are normally GGTase I substrates rescued proliferation
defects caused by deletion of the gene encoding GGTaseI
in mammalian cells, suggesting that these proteins can
be modified by FTase under these conditions41. This
phenomenon of cross-prenylation has received the most
attention with respect to its potential to affect CAAX
protein function when FTase inhibitors are used thera
peutically. Although they normally undergo farnesyl
ation, two RAS isoforms KRAS and NRAS can
also be processed by GGTaseI, and geranylgeranylation
of these RAS proteins can be detected in human cancer
cells when FTase is inhibited42. This cross-prenylation
process is thought to be at least partially responsible for
the resistance of some RAS-mutant cancers to treatment
with FTase inhibitors6,43. Mammalian proteins that can be
modified by either isoprenoid under physiological conditions have also been identified; the most well-studied
example is the GTPase RHOB44,45. The differently prenyl
ated forms of RHOB seem to have unique roles in cell
signalling 46,47, which adds another interesting dimension
to the r egulatory role of prenylation.
Until recently, there had been little information
regarding the potential regulation of prenylation.
However, recent studies have shown that splice variants of SMGGDS, which are nucleotide exchange factors for some small GTPases, have a second function in

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regulating the prenylation and trafficking of their target
GTPases48. There are two variants of SMGGDS, named
SMGGDS607 and SMGGDS558, which recognize different Cterminal structural features of CAAX proteins.
SMGGDS607 preferentially binds to small GTPases
containing a polybasic region (PBR) upstream of the
CAAX motif soon after synthesis in their unprenylated
form, and this seems to facilitate their recognition by
GGTase I. SMGGDS558, by contrast, binds to PBRcontaining GTPases after prenylation by GGTase I,
facilitating post-prenylation processing and targeting
to the plasma membrane49. Although much remains
unknown about the functions of SMGGDS, the process clearly represents a novel facet of the regulation of
prenylation and the trafficking of CAAX proteins50.
Post-prenylation processing by RCE1 and ICMT. The
protease responsible for the maturation of CAAX proteins, RCE1, was originally identified in a screen to
detect genes involved in the processing of yeast Ras
proteins30. Its mammalian counterpart, also termed
RCE1, was identified shortly afterwards, on the basis of
its homology to the yeast gene51. Both yeast and human
RCE1 are the post-prenylation proteases for almost
all of the CAAX proteins in both species. Another
yeast protease, sterile 24 (Ste24; also known as Afc1)
is involved in the post-prenylation proteolysis of yeast
a-factor only; its mammalian orthologue, zinc metallo
proteinase Ste24 homologue (ZMPSTE24), is involved
in a distinct proteolytic processing of the prelamin A
protein (seebelow)5255.
Similarly to RCE1, the enzyme responsible for carboxyl methylation of CAAX proteins was also first
identified in yeast and was named Ste14 (REF.32). The
mammalian orthologue was identified later and is now
known as ICMT31,56. It is intriguing that both ICMT and
RCE1 reside on the endoplasmic reticulum (ER) rather
than on the plasma membrane, which is the functional
destination for many CAAX proteins. The prevailing
view is that newly prenylated proteins in the cytosol
rapidly encounter RCE1 and ICMT on the cytoplasmic
face of the ER, unless they are sequestered somehow
(for example, by binding to SMGGDS), which suggests
that the two steps of post-prenylation processing are
efficiently coordinated.
RCE1 and ICMT can modify both farnesylated and
geranylgeranylated CAAX proteins. As they are potential
drug targets5, it would be advantageous to have detailed
structural information on these proteins. However, as
they are polytopic membrane proteins that are difficult
to solubilize while retaining enzymatic activity, their
biochemical characterization has lagged behind that
of FTase and GGTase I. A prokaryotic orthologue of
RCE1 has been crystallized, and the data indicate that
it uses a glutamate-activated water molecule for catalysis57. The same group has also solved the structure of a
prokaryotic orthologue of ICMT58. As prokaryotes contain no recognized prenylated proteins, it is not clear
how closely these gene products functionally resemble eukaryotic RCE1 and ICMT, particularly in terms
of the prenylpeptide binding sites. Structurefunction
studies of mammalian and yeast ICMT have provided

some insights into its orientation within the ER membrane and have identified amino acids that are crucial
for its catalytic activity 59,60. A recent study suggests
that ICMT may function as a homodimer 61. Similarly,
structurefunction analyses of RCE1 have yielded data
with regard to its membrane orientation and the residues
that are important in catalysis62,63. For both RCE1 and
ICMT, the mutagenesis results have pointed to residues
both in transmembrane regions and in predicted cyto
plasmic loops as being important in catalysis, reinforcing
the notion that RCE1 and ICMT process CAAX proteins
on the cytoplasmic face of theER.
Biological roles of prenylation

Protein trafficking. The combination of the unique
structure of each CAAX protein and the prenylation
modification endows each protein with a characteristic
pattern of membrane trafficking and cellular localization
that is crucial for its function. Although most studies
examining the biological effects of loss of prenylation
have focused on the impaired membrane attachment of
the CAAX proteins, there are several examples in which
the attached lipid is involved in crucial proteinprotein
interactions. Some of the best-characterized roles of
protein prenylation in proteinprotein interactions
involve subcellular trafficking of CAAX proteins (FIG.2).
Trafficking between different membrane compartments,
and between membrane compartments and the cytosol,
is an integral part of the life cycle of most prenylated
proteins, as they are modified first in the cytosol and
then on the ER, whereas their sites of function often
involve other cell membranes. The trafficking process
can be facilitated by several binding or chaperone proteins. For RHO GTPases, the geranylgeranyl modification is a crucial determinant of their interaction with
a group of cellular proteins, termed RHOGDIs, which
influence the localization, GTP-binding properties and
activities of RHO family members64,65 (FIG.2a). This
binding of RHOGDIs to distinct RHO isoforms may
also be influenced by modifications such as palmitoyl
ation at or near the prenylated cysteine66. For the RND
subfamily of RHO GTPases, a recent report showed that
1433 scaffold proteins interact with a hybrid prenyl-
phosphorylation motif on the RND protein to inhibit the
activity of these GTPases67. Direct structural evidence
shows that both RHOGDI and 1433 proteins have
hydrophobic pockets that accommodate the isoprenoid
moiety of RHO GTPases64,67.
In addition to the extensively investigated interaction
of RHO proteins with prenyl-interacting proteins, the
interaction of RAS proteins with a prenyl-binding protein termed phosphodiesterase- (PDE) has generated
interest 68. PDE regulates the membrane release and
distribution of RAS and some other farnesylated proteins (FIG.2b). PDE and RHOGDI have similar structures and have related functions that involve binding to
prenylated proteins and transiently sequestering them
in the cytosol as part of a regulated process to control
the localization, and therefore the function, of these
proteins. The functional relevance of this interaction
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a
Plasma membrane
RAC
GTP
CDC42
GTP
Pi
RHOA
GTP
RAC
GDP
CDC42
GDP
Competition
RHOA for RHOGDI
GDP
RHOA
GDP
Extraction
GEF
Eector
GTP
GDP
RHOGDI
Drop-o
RAC
GDP
Signalling
CDC42
GDP
Drop-o
RHOA
GDP
Extraction
RHOGDI
RHOA
GDP
RHOA
GDP
ICMT
RHOA
GDP
RCE1
Ribosome
RAC
GDP
CDC42
GDP
GGTase
RHOA
GDP
ER membrane
RHOA
GDP
b
PAT
N RAS
H
N
H RAS
Ca2+
+
Plasma
membrane
PPT
RAS
RAS
CaM
Vesicular
tracking
+
ARL2/3
PRA1
GTP
Pi
++ +
KRAS
++ +
KRAS
Galectin
RA
GT S
P
PAT
PDE
ARL2/3
GDP
GEF
GAP
ARL2/3
GTP
PDE
RAS
PPT
RAS
KRA
Intracellular
membrane
Signalling
Nature Reviews | Molecular Cell Biology

Figure 2 | Roles of protein-associated isoprenoids in proteinprotein interactions
and membrane targeting.
a|Geranylgeranyl-dependent binding of RHOGDI to GDP-bound RHO GTPases regulates their activities, distribution
and stability. The aminoterminal domain of RHOGDI preferentially binds to the switch domain of the GDP-bound
RHOA, RACor CDC42, and the hydrophobic pocket in the carboxyterminal domain of RHOGDI extracts the prenyl
group of the GTPase from the membrane. This binding locks RHO in its GDP-bound form, thereby reducing the pool of
both membrane-associated and activated forms of RHO. Through this mechanism, RHOGDI balances the functions and
effects of different RHO family members under diverse physiological conditions. b | Prenylation has roles in the binding
of RAS GTPase to interacting proteins and in membrane trafficking and signalling. All RAS isoforms travel between
intracellular membranes such as the endoplasmic reticulum (ER) and the plasma membrane at some point during their
life cycle. A diverse array of interacting proteins has been implicated in the transport of different RAS GTPases.
Phosphodiesterase- (PDE) and prenylated RAB-acceptor protein 1 (PRA1) function as regulators for the solubilization
and membrane distribution of RAS, as well as some other processed farnesylated proteins. PDE, which is the better
studied of the two, binds and removes from the membrane the prenylated (but preferably unpalmitoylated) RAS
proteins, independently of their GTP/GDP-binding status. This process is subject to regulation by the ADP-ribosylation
factor-like protein 2 (ARL2) or ARL3 (ARL2/3) GTPases. Only the GTP-bound form of ARL proteins can bind to PDE, and
the binding of either ARL-GTP orprenylated RAS to PDE is mutually exclusive; hence, the PDE-mediated solubilization
of RAS is regulated by the GTP/GDP cycling of ARL. Calcium-activated calmodulin (CaM) can interact with both farnesyl
lipid and the polybasic region of KRAS, facilitating its extraction from the plasma membrane and triggering cytosolic and
endomembrane redistribution; this has been reported to contribute to differential KRAS signalling. Another family of
proteins, the galectins, are reported to bind to activated RAS proteins through interaction with the farnesyl lipid and an
adjacent region of RAS, and to stabilize their membrane attachment and enhance signalling. GAP, GTPase-activating
protein; GEF, GDP/GTP exchange factor; GGTase, protein geranylgeranyltransferase; ICMT, isoprenylcysteine
carboxylmethyltransferase; PAT, palmitoyl acyltransferase; PPT, palmitoyl protein thioesterase; RCE1, RAS-converting
CAAX endopeptidase 1.

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between PDE and prenylated RAS proteins has been
highlighted by thefinding that small molecules that
selectively bind tothe prenyl-binding pocket of PDE
impair its interaction with KRAS and inhibit oncogenic
signalling 69. Unlike RHOGDI, which interacts only with
geranylgeranylated RHO family proteins, PDE interacts
with a broad range of farnesylated proteins, including all
RAS family members, as well as RHEB and certain RAP
proteins. This difference in binding specificity and flexi
bility can be explained by the structural determinants
ofbinding. Unlike RHOGDI, which has multiple sitesof
contact with the RHO protein, PDE binds almost
exclusively to the Cterminal farnesylated motif, which
accounts for the indiscriminate nature of the interactions
of PDE with both GTP- and GDP-bound proteins.
Interestingly, the binding of PDE to RAS seems to be
regulated by the GTPases ADP-ribosylation factor-like
protein 2 (ARL2) and ARL3, depending on their GTPbinding status70. Other proteins that have been implicated in controlling the cellular distribution of RAS
through interactions with its prenylated C terminus are
galectins71, calmodulin72 and prenylated RAB-acceptor
protein 1 (PRA1)68,73 (FIG.2b).
FTase inhibitors
(FTIs). Inhibitors of the protein
farnesyltransferase (FTase)
enzymes. FTIs have been used
in clinical trials for the
treatment of cancer, progeria
and hepatitis D virus infection.
Cellular roles of protein prenyltransferases. In addition

to being the first form of protein prenylation to be discovered, farnesylation has attracted attention because
of the important role of the farnesylated protein RAS
in carcinogenesis74. Although it is now recognized that
NRAS and KRAS can also be geranylgeranylated, and
that RAS family members are not the only CAAX proteins that have active roles in cancer and other patho
logical conditions3,75, much of our knowledge of the roles
of protein prenylation in modulating the functions of
CAAX proteins was obtained during the quest to understand the effects of FTase inhibition on RAS membrane
association and signalling.
Suppression of FTase activity has complex and cell
context-dependent anti-proliferative effects. Genetic
inactivation of FTase in mice blocked the proliferation
of fibroblasts, and conditional inactivation of FTase in
KRAS-induced lung cancer in mice markedly impaired
tumour growth and improved survival76. FTase inhibitors
(FTIs) can induce G1 phase and G2M phase cell cycle
arrest in different types of cancer cell, effects that are not
always linked to the status of RAS activation7,77. In the
case of cancer cells with mutations in KRAS and NRAS,
the effects of FTIs on the cell cycle and proliferation correlate poorly with changes in the prenylation status of
RAS proteins, which suggests that other CAAX proteins
have roles in the regulation of cell proliferation. Indeed,
centromere-associated protein E (CENPE) and CENPF
have been identified as farnesylated proteins that partici
pate in cell cycle progression at the mitotic phase78,79.
Nuclear lamins also comprise a group of farnesylated
proteins that have been implicated in both cancer and
ageing, and evidence suggests that FTIs disrupt the processing and localization, and presumably the function,
of lamin A/C and lamin B80,81. Two farnesylated substrates, RHEB GTPase and liver kinase B1 (LKB1; also
known as STK11), are involved in the control of mTOR
and AMP-activated protein kinase (AMPK), which are

central sensors and regulators of cellular energy metabolism82,83. Although the details of the mechanism are under
investigation, it is likely that their CAAX processing is
functionally relevant84.
Although the development of FTIs is more advanced,
owing to the prominent role of RAS in tumorigenesis,
several cell-active inhibitors of GGTase I (GGTIs) have
been described8588. As there are more predicted geranyl
geranylated CAAX proteins than farnesylated proteins in
the mammalian proteome24,25, the mechanisms of the biological effects of GGTI treatment are even more difficult
to delineate than those of FTI treatment. Nonetheless,
studies with GGTIs have revealed several biological consequences of suppressing protein geranylgeranylation.
Administration of GGTIs to cells generally causes cell
cycle arrest at G0G1, which is thought to be mediated by the inactivation of cyclin-dependent kinase2
(CDK2) and CDK4 by the p21 and p15 kinase inhibitors
downstream of RHO89. Suppression of GGTaseI also
potently stimulates apoptosis, through a mechanism
that is believed to involve both PI3KAKT and survivin
pathways90,91. RHO family GTPases are the best studied
in terms of mediating the effect of GGTIs on cell cycle
regulation and cell migration85,86. Itis also important to
note that the effect of GGTIs may be mediated, at least
partially, through CAAX proteins such as RHOB, which
can be modified by both GGTaseI andFTase.
Impact of RCE1mediated proteolysis. Genetic disruption
studies in mice indicate that RCE1 is the only enzyme
that can proteolytically process the majority of CAAX
proteins53. Removing the three Cterminal residues
of prenylated CAAX proteins is essential for development; mice lacking RCE1 die late in gestation or within
the first week of life, with no apparent organogenesis
defects53. Post-development phenotypes have also been
studied following deletion of RCE1 in specific tissues,
most notably in the heart; mice with cardiac deletion
of Rce1 developed dilated cardiomyopathy at an early
age and died by 10months92. Although the CAAX protein(s) responsible for the consequences of limiting
RCE1 activity have not been identified, it seems likely
that there are tissue-specific effects of RCE1 dysfunction
and that some substrates of RCE1 will be more affected
by incomplete processing than others. This notion is
supported by a recent study in which Rce1 was specifically deleted in the retina; the ensuing retinal degeneration was shown to be linked to defective transport of
one prenylated protein, PDE6, from the cell body to the
outer segments of photoreceptor cells, and the transport
of other prenylated proteins (such as transducin) in the
retina wasunaffected93.
Impact of ICMT-catalysed methylation. Gene disruption
studies have indicated that ICMT is the only enzyme that
catalyses the carboxyl methylation of prenylated proteins.
Icmt is an essential gene in mouse development; animals
that lack Icmt die around embryonic day 11.5, although
few defects have been reported other than a modest
defect in liver development 94,95. It seems surprising that
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GDP/GTP exchange factors

(GEFs). Proteins that facilitate
the exchange of GDP for GTP in
the nucleotide-binding pocket
of a GTP-binding protein.
mice lacking ICMT die earlier in development than those

lacking RCE1, as RCE1 functions before ICMT in the
processing of CAAX proteins. One possible explanation
comes from the finding that ICMT can methylate a subset of prenylated RAB proteins in addition to prenylated
CAAX proteins94,96. Another possibility is that, for certain
proteins, the AAX extension on the prenylcysteine may
be better tolerated than a negatively charged unmethylated carboxylate for membrane association, interaction
with regulatory proteins, orboth.
ICMT inhibition led to G1 cell cycle arrest and cell
death in many types of cancer cell, similar to the effects of
FTIs and GGTIs. One finding that was not predicted by
earlier studies of the prenyltransferase inhibitors was that
suppression of ICMT promoted robust autophagy and cell
death linked to autophagy, which might be a mechanism
for the antitumour effects of ICMT inhibition97. These
findings provided the first link between protein prenyl
ation and the process of autophagy, a connection that
has now also been observed using FTIs and GGTIs98,99.
Although the mechanism is unclear, it is likely that multi
ple ICMT substrates, including RAC3 GTPase, regulate
autophagy 100. Autophagy is an evolutionarily conserved
cellular response to an energy-depleted state, and as
such it is subject to complex regulation101,102. Arecent
study showing that ICMT inhibition suppresses mito
chondrial respiration, leading to an energy-depleted state
and an increase in autophagy 103, has provided additional
insight into this process. Experiments with both isogenic
cells lacking mitochondria and agents thatinhibit the
electron-transport chain demonstratedthat the suppression of oxidative phosphorylation can account for much
of the growth inhibition observed following treatment
with an ICMT inhibitor. Further investigation to identify the CAAX protein(s) involved in the regulation of
mitochondrial function will be essential to understanding the cellular function of ICMT and for the possible
therapeutic application of ICMT inhibitors.
Early studies provided evidence that methylation is
important for the function of RHO GTPases104,105. One
of the main functions of RHO proteins is to tightly
coordinate rearrangement of the actin cytoskeleton and
cellpolarization in response to stimuli that modulatecell
migration and invasion106. Cell migration is necessary for
development and for many normal cellular functions,
and dysregulation of this process is a driving element in
cancer metastasis107. In a recent study assessing the effect
of ICMT on cell-biological processes associated with
the function of RHO proteins, inhibition of ICMT triggered disruption of the actin cytoskeleton and impaired
ligand-mediated activation of RHOA and RAC1
(REF.108). This effect was linked to a tighter binding of
the regulatory molecule RHOGDI to both RHOA and
RAC1 in their unmethylated states, which presumably
impaired their ability to be activated by GDP/GTP exchange
factors (GEFs)108,109. Cterminal methylation also affects
the stability of RHO proteins, with different family members behaving differently in this regard. The half-life of
methylated RHOA is markedly longer than that of the
unmethylated protein104,105, whereas it is the opposite for
RHOB, suggesting that additional structural elements are
involved in the degradation of these proteins110. RHOA

and RHOB have distinct cellular functions, and it is possible that this differential regulation of stability by ICMT
coordinates these functions. In any event, it seems that
ICMT function can affect the activities of its substrate
proteins by influencing steady state proteinlevels.
CAAX protein methylation is the only potentially
reversible step in the process of protein prenylation,
which highlights the regulatory importance of this step.
There is evidence in support of the dynamic methylation
of CAAX proteins having regulatory consequences. For
example, large pools of unmethylated RHO proteins have
been observed in both endothelial111 and breast cancer112
cells, and manipulating the levels of unmethylated versus
methylated RHO affected RHO-dependent processes in
the cells. It has long been hypothesized that there might
be esterases that can demethylate prenylcysteines104,111,
and recent studies support this113,114. Direct evidence
came from the recent identification of a carboxylesterase,
CES1, which has a marked effect on the methylation status of RHO104. Notably, CES1 silencing in breast cancer
cells affected RHO function and cytoskeletal organization in a manner similar to ICMT overexpression, which
shows that dynamic methylation of RHOA can affect its
physiological function. Furthermore, an orthologue of
CES1 had previously been identified in plants as having
prenylcysteine carboxylmethylesterase function, and as
being involved in a signalling pathway that is important
in meristem development 115. These findings open an
exciting new avenue of research for understanding the
physiological importance of Cterminal methylation
ofproteins.
Targeting CAAX protein processing in disease

Manipulation of the protein prenylation pathway has
been suggested as a therapeutic approach for several
human diseases3,7. The main areas in which these efforts
have been focused are summarizedhere.
Cancer and inflammation. RAS is the most well-studied
driver oncogene in tumour initiation and maintenance74.
RAS has been notoriously difficult to target directly,
owing to its high affinity for GTP and the similarities of
its core functional domain with those of other GTPases116.
Hence, the discovery of the essential role of farnesylation
in modulating RAS function generated enormous enthusiasm for the development of FTIs117. The impressive
antitumour activity and low toxicity of FTIs observed in
animal models quickly led to clinical trials, but the results
of these trials have been disappointing 118. Encouraging
responses have been observed in haematological cancers
and some aggressive breast cancers119, but the overall
response of patients is poor 120. As noted above, the alternative prenylation of KRAS and, to a lesser extent, of
NRAS by GGTase I has been invoked as a potential
mechanism by which tumour cells can escape the effect of
FTIs. In support of this, tumours that are driven by mutation of HRAS, which can only be farnesylated, are sensitive to FTIs. For example, early studies with transgenic
mouse models of HRAS-mutant mammary and salivary
carcinomas showed FTI-dependent tumour regression121,

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and a recent study using a mouse model of HRAS-mutant
papilloma and other human cancer cell lines illustrated
the efficacy of FTI treatment in HRAS-driven, but not
KRAS- or NRAS-driven, cells122. However, it is likely that
there are other mechanisms of FTI escape in addition
to alternative prenylation. The identification of predict
ive biomarkers for FTI efficacy would greatly aid in the
development of FTIs as therapeutic agents for cancer 120.
The recognition of alternative prenylation of RAS
enhanced attention on targeting GGTase I. A genetic
study has shown that conditional deletion in myeloid and
lung cells of the gene encoding the unique subunit of
GGTase I almost completely eliminated the proliferation
and tumour formation that accompanied the induction
of oncogenic KRAS expression, with markedly improved
survival of the mice41. A further study showed that concurrent genetic inactivation of both FTase and GGTaseI
had a stronger effect in terms of reducing lung cancer
burden and increasing mouse survival, compared with
single-enzyme inactivation76. Proteins solely processed by
GGTase I for example, RHO proteins are involved
in various pathologies, including cancer and inflammation. Hence, in addition to their potential to suppress the
alternate prenylation of KRAS and NRAS and thus affect
KRAS- and NRAS-dependent oncogenesis(BOX2), GGTIs
Box 2 | Insights from the use of statins
Inhibitors of HMG-CoA reductase (HMGCR), most notably the statin group of small
molecules, are potent lipid-lowering agents that are best known for their effects on
cholesterol synthesis. However, inhibition of HMGCR does not only affect sterol levels.
The direct product of HMGCR, mevalonate, is the precursor of farnesyl diphosphate
(FPP), which can be elongated to geranylgeranyl diphosphate (GGPP) or cyclized to
produce squalene, the first committed intermediate in the production of sterols.
Squalene production is the most sensitive to limiting levels of FPP, followed by GGPP
synthesis, with protein farnesylation by farnesyltransferase (FTase) being the least
affected by HMGCR inhibition, owing to the different binding affinities of the
FPP-utilizing enzymes of each pathway (namely, squalene synthase, GGPP synthase and
FTase)36,161,162. Statins are commonly used worldwide, which has allowed large population
studies of individuals who have taken the drug for an extended period of time.
Thesestudies have led to the identification of beneficial effects that do not seem to be
aconsequence of reduced cholesterol levels. Disorders that have been reported to occur
less frequently in individuals on long-term statin therapy include stroke, some chronic
inflammatory disorders, osteoporosis and several types of cancer163. Statins have also
been found to promote non-amyloidogenic processing of the amyloid precursor protein
and to reduce the production of amyloid -protein (A) that has been implicated in the
development of Alzheimer disease154,164.
Not all of the consequences of statin treatment are positive. It has long been known that
a subset of individuals taking statins experience muscle damage, which can be severe165.
Most of these outcomes are thought to be independent of the cholesterol-lowering
effect of statins; instead, they seem to be due to a reduction in cellular isoprenoid levels.
For this reason, much attention has been focused on the geranylgeranylisoprenoid,
GGPP, and on protein geranylgeranylation. Estimates of flux through the isoprenoid
biosynthetic pathway suggest that, at limiting isoprenoid levels, protein farnesylation
would be preserved at the expense of geranylgeranylation5. Consistently, in cellular
models of statin-induced isoprenoid deprivation, most of the sterol-independent
consequences for example, proliferation, migration and self-renewal of stem cells
can be reversed by adding geranylgeraniol, which is a precursor of GGPP, to the
media162,166. In terms of specific geranylgeranylated proteins that might contribute to
themultiple biological effects of GGPP depletion in cells, the majority of reports focus on
theRHO family of GTPases167,168. Most recent in this regard is the finding that isoprenoid
biosynthesis specifically, GGPP production and RHO activation are required for
engagement of the YAPTAZ axis in the Hippo signalling pathway, and that this process
enables the proliferation and self-renewal of breast cancer cells169,170.
have been proposed as potential therapeutics in RASindependent diseases123,124. GGTIs have been investigated
for use in anti-angiogenic therapy 125, as agents against the
metastatic progression of cancers (probably through an
effect on proteins such as RHOA and RHOC)126, and as
agents for inflammatory disorders127. So far, GGTIs have
shown significant efficacy in several animal models of
both cancer and inflammatory diseases, and one such
agent (known as GGTI2418) has gone through PhaseI
clinical trials7.
There is much that we do not yet understand regarding the effects of GGTase I on the function of CAAX proteins in different cellular contexts. The effects of GGTaseI
inhibition on cellular processes associated with inflammation are particularly complicated, as exemplified
bythe surprising result from a recent study examining
the effects of conditional deletion of the gene encoding
GGTase I in macrophages128. The expectation had been
that inactivation of the enzyme would provide some protection from inflammatory symptoms, on the basis of the
pro-inflammatory effects of RHO GTPases106,129. Instead,
the mice developed severe joint inflammation that resembled rheumatoid arthritis, which was suggested to be due
to the paradoxical activation of RHO family members in
the absence of modification by GGTase I128. These studies
not only demonstrated the complex regulation of RHO
activities by GGTase I, but, more importantly, they highlighted the cell context-specific interplay between signalling pathways in determining the biological processes
influenced by CAAX protein geranylgeranylation.
From a cancer therapy perspective, the post-
prenylation processing steps are attractive targets; RCE1
and ICMT modify both farnesylated and geranylgeranyl
ated proteins, and therefore their inhibition could affect
the function of oncoproteins that are modified by either
prenyltransferase. Genetic studies show that Rce1 deletion has relatively limited consequences compared with
deletion of the protein prenyltransferases. For example,
although conditional deletion of Rce1 in fibroblasts did
reduce RAS-induced transformation of the cells130, the
effect was small compared with that of targeting FTase
or GGTase I. If agents targeting RCE1 are developed for
clinical use, either as a single agent or in combination
with other prenylation inhibitors, it will be important
to evaluate their safety carefully, given the findings that
cardiomyopathy and retinopathy can occur upon targeting the enzyme in mice92, and that KRAS-driven myeloproliferative disease is enhanced upon RCE1 deletion in
haematopoietic cells131.
In contrast to the modest effects of RCE1 disruption
on oncogenesis, eliminating ICMT activity has profound
consequences in several systems. In the first such study,
the ability of oncogenic KRAS to transform immortalized fibroblasts was eliminated by conditional deletion
of Icmt95. Interestingly, ICMT deletion also attenuated
transformation by activated RAF kinase, which highlighted the importance of CAAX proteins in addition to
RAS in oncogenesis, and the essential role of carboxyl
methylation for the functions of these CAAX proteins.
In a subsequent mouse model, conditional inactivation
of ICMT reduced the development of lung tumours and
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a Normal prelamin A processing
b Abnormal prelamin A processing

ZMPSTE24 loss of function
FTase
FTase
CAAX
RSYLLG
RCE1
COO
ICMT
RSYLLG
RSYLLG
Toxic progerin
(prenylated and
methylated prelamin A)
RSY
COCH3
CAAX
RCE1
del
ICMT inhibition
ICMT
ZMPSTE24
del
COO
CAAX
FTase
CAAX
RCE1
RSYLLG
COCH3
del
CAAX
FTase inhibition
RSYLLG
AA
AA
RSYLLG
RSYLLG
CAAX
AA
RSYLLG
HGPS (lack of ZMPSTE24

cleavage site)
COO
ICMT
del
COCH3
Toxic progerin
(prenylated and
methylated prelamin A)
LL
CH
CO
Mature lamin A
Figure 3 | Completion of prelamin A maturation into lamin A depends on threestep

prenylation
processing.
Nature Reviews
| Molecular
Cell Biology
a|Innormal prelamin A processing, following farnesylation by farnesyl transferase (FTase), RAS-converting CAAX
endopeptidase 1 (RCE1)mediated proteolysis and isoprenylcysteine carboxylmethyltransferase (ICMT)-mediated
carboxyl methylation, the ZMPSTE24 protease cleaves the carboxyterminal 15 residues from prelamin A to produce
mature lamin A. b | Abnormal prelamin A processing results in the accumulation of progerin. The most well-studied
abnormalities in prelamin A processing associated with disease are the result of loss-of-function mutations of the gene
encoding the ZMPSTE24 protease or, in patients with HutchinsonGilford progeria syndrome (HGPS), point mutation of
the prelamin A gene that results in a deletion in the amino acid sequence spanning the ZMPSTE24 cleavage site. In both
cases, toxic progerin accumulates, resulting in the manifestation of premature ageing phenotypes in the affected
organisms. Inhibition of FTase or ICMT shifts the pool of prelamin A to the unfarnesylated or uncarboxymethylated form,
respectively, both of which presumably have reduced toxicity compared with progerin.
Progeroid syndromes
A group of rare genetic
disorders that result in
markedly accelerated ageing.
myeloproliferation phenotypes induced by concurrent

activation of KRAS in the same cell population132. By
contrast, a recent study has shown that Icmt deletion
exacerbated disease progression in a mouse model of
KRAS-driven pancreatic cancer 133. It is worth noting
that in this system, mice were engineered such that
KRASG12D expression and ICMT inactivation would
simultaneously occur in embryonic pancreatic tissue.
The consequences of the loss of ICMT function during
early development would probably not be the same as
those of suppression of ICMT in pancreatic tumours
post-development. Itwould be interesting to see whether
the same exacerbation of disease would occur if ICMT
was deleted post-development, preferably following the
activation of KRAS and tumour initiation. These findings underscore the need to better understand the role
of prenylation, particularly the methylation step, in the
context of d
evelopment as well as tumorigenesis.
Several groups have identified small molecules that
potently inhibit ICMT enzymatic activity 134137. One such
inhibitor, termed cysmethynil, has shown activity both
in a variety of cancer cell lines invitro and in multiple

human cancer xenograft models invivo97,134,138. Evidence
from various approaches, including second-generation
compounds with increased potency and biological
activity 139, indicates that ICMT inhibition causes cell
cycle arrest, autophagy and cancer cell death. A clue to
the mechanism by which autophagy is induced comes
from the finding noted above that ICMT inhibition profoundly impairs mitochondrial oxidative phosphoryl
ation103. Further investigation of the role of ICMT in the
regulation of cell respiration and in tumorigenic processes will be important for the clinical development of
ICMTinhibitors.
Ageing. There is considerable interest in targeting protein
prenylation in premature ageing disorders and, potentially, in normal ageing 133,140. In cell and mouse models of progeroid syndromes, which are characterized by
abnormal processing of the CAAX protein prelaminA
and its subsequent accumulation, disease progression
can be substantially alleviated by inhibiting prenylation

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processing 141,142. The maturation of lamin A begins in a
similar manner to that of a typical farnesylated CAAX
protein, with sequential threestep modifications.
However, the final maturation step involves the removal
of the Cterminal 15amino-acid peptide by the endoprotease ZMPSTE24, which is dependent on prenyl
ation processing 143 (FIG.3a). The most well-studied form
of the disorder, HutchinsonGilford progeria syndrome
(HGPS), is caused by a genetic mutation of prelaminA
that leads to alternative splicing, with the consequent
loss of the ZMPSTE24-recognition site. A more severe
formof progeria is caused by the loss of ZMPSTE24 protease activity through genetic mutation144146 (FIG.3b). In all
forms of progeria, the lack of proteolysis of farnesylated
and carboxymethylated prelamin A results in the accumulation of an incompletely processed molecule, termed
progerin, on the nuclear envelope, where it triggers a
range of molecular disturbances that lead to premature ageing 10,147. Interestingly, FTI treatment reverses
the phenotypical abnormalities in progeroid cells141,148,
and knockin of a form of prelamin A that could not
be farnesylated, on the genetic background of progeria,
yielded mice that had relatively normal survival149. These
studies led to a clinical trial using FTIs to treat progeroid
syndromes. Although FTIs had only a modest effect on
disease progression141, the results prompted an ongoing
trial using a combination of three agents that affect the
prenylation pathway a statin, a bisphosphonate and
a FTI and early data have shown that the triple-drug
combination can reduce the multi-organ phenotype of
ageing and significantly prolong survival150. Progress
has also been made in investigating the effects of targeting ICMT in progeroid disorders; introduction of a
Box 3 | Protein prenylation in parasite biology and viral assembly

Protein prenylation occurs in a wide range of parasites, including Trypanosome spp.,
Leishmania spp., Plasmodium falciparum and Giardia lamblia, and farnesylation seems
to be the dominant form of the modification in these organisms171. Many parasites are
exquisitely sensitive to impairment of protein prenylation. These findings have led to
the use of protein farnesyltransferase (FTase) inhibitors (FTIs) that were originally
developed as anticancer agents in protozoan parasitic diseases171. In addition, the FTase
in parasites is sufficiently distinct from the mammalian FTase that it is possible to
develop FTIs that are selective for the parasite enzyme172.
Protein prenylation is also exploited by viruses. The first example of this process being
important in viral replication came from the discovery that the hepatitis D virus (HDV)
large antigen is a CAAX protein173 that is processed by the host FTase174. Genetic or
pharmacological suppression of prenylation of the HDV large antigen abolishes its
ability to form virus-like particles with the hepatitis B virus (HBV) surface antigen175.
Therequirement for coinfection with HBV results in HDV infection being the most
fulminant form of viral hepatitis in humans; therefore, the potential of controlling the
disease with prenylation inhibitors is considered to be a clinically important area of
research176. Consequently, in 2014, orphan designation was granted by both the US
Food and Drug Administration (FDA) and the European Commission for the use of the
FTI lonafarnib in the treatment of HDV infection, which was followed shortly after by a
report of its efficacy in a clinical trial177. In addition to viral proteins that directly
undergo isoprenoid modification, host prenylated proteins can have crucial roles in viral
assembly. This was first noted when inhibition of protein geranlygeranyltransferaseI
(GGTase I) was found to trigger disassembly of hepatitis C virus (HCV) replication
complexes156. This effect has been attributed to a host CAAX protein, named F-box and
leucine-rich repeat protein 2 (FBL2). FBL2 is processed by GGTase I, and its association
with the HCV protein NS5A is required for HCV replication178.
hypomorphic allele of Icmt into the Zmpste24 / mouse

model of progeria largely rescued both the premature
ageing phenotype and premature death151.
Other pathologies. Several studies have demonstrated
that specific GTPases, including members of the RAS
and RHO subfamilies, are important players in the regu
lation of synaptic plasticity 152. Furthermore, a recent
study in a neuronal cell model has shown that statins
affect neuron growth cone and neurite outgrowth in
a geranylgeranyl-dependent manner, suggesting the
involvement of geranylgeranylated proteins in these
processes153. There is growing interest in understanding
the effects of prenylation on neurodegenerative disorders,
with findings that suggest prenylated proteins may have
important roles in the pathogenesis of Alzheimer disease154. Of interest in this regard is a recent study showing
that haploinsufficiency of FTase reduced neuropathology
and rescued cognitive function in a mouse model of
Alzheimerdisease155.
Another area in which research attention has been
focused is infectious diseases. Hepatitis C virus (HCV)
replication can be attenuated by inhibiting geranyl
geranylation156, indicating that the replication of HCV in
liver cells requires a GGTase I substrate. Targeting prenylation is also a strategy to combat hepatitis D virus (HDV)
and several tropical diseases (BOX3).
Conclusions and perspectives

Since the discovery that Cterminal processing through
the prenylation pathway was required for oncogenic
forms of RAS to transform cells, elucidating the mechanisms and consequences of CAAX protein processing
has been a major area of study. Attention was initially
focused on the prenyltransferases FTase and GGTaseI
owing to the marked consequences of blocking the
lipid-attachment step of protein prenylation. The
identification of the mammalian genes encoding RCE1
and ICMT, followed quickly by reports that disruption of these genes in mice had major developmental consequences, has highlighted the importance of
understanding how all of the prenylation-dependent
processing steps contribute to the biological activity of
CAAXproteins.
After more than two decades of investigation, we
now have a good understanding of the biochemical processes of prenylation and the enzymes that are involved.
The focus has shifted to elucidating the effects of each
processing step on the activities of individual CAAXproteins. In this regard, it is key to identify the CAAX
proteins that are involved in the antitumour response
to FTIs, GGTIs and ICMT inhibitors. Answering these
questions is complicated, not only because many regulatory pathways (and many pathological processes) involve
multiple CAAX proteins, but also because the function of
each CAAX protein must be placed into a particular cellular context. The notion that a strategy to attenuate, but
not necessarily eliminate, the function of CAAX proteins
might prove effective in targeting cancer and potentially other diseases, as discussed above makes these
challenges worthy of attention.
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Acknowledgements
The authors thank the reviewers for helpful comments on the

manuscript and express regret that they could not reference
all of the important studies that have contributed to the
understanding of protein prenylation. They also thank Zheng
Huan Tay for his assistance with the figures. Work from the
authors laboratories is funded by the Ministries of Health
and Education of Singapore.
Competing interests statement
The authors declare no competing interests.


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REVIEWS

Protein prenylation: unique fats make

Abstract | The modification of eukaryotic proteins by isoprenoid lipids, which is known as

Program in Cancer and Stem

Life would have been so much simpler for molecular and

Prenylation provides proteins with a hydrophobic

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

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RAS CCOO CH3

Figure 1 | Isoprenoid metabolism and CAAX protein prenylation. a | The production

the maturation of prelamin A depends on prenylation,

A brief history of protein prenylation

in a way that could not be reversed by adding sterols14,15

2 | ADVANCE ONLINE PUBLICATION

Thesubsequent discoveries that human RAS proteins

The three enzymatic steps of protein prenylation

is synthesized downstream of mevalonate (FIG.1). Both

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

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studies of mammalian and yeast ICMT have provided

Biological roles of prenylation

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Nature Reviews | Molecular Cell Biology

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Cellular roles of protein prenyltransferases. In addition

and AMP-activated protein kinase (AMPK), which are

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GDP/GTP exchange factors

mice lacking ICMT die earlier in development than those

involved in the degradation of these proteins110. RHOA

Targeting CAAX protein processing in disease

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8 | ADVANCE ONLINE PUBLICATION

b Abnormal prelamin A processing

HGPS (lack of ZMPSTE24

Figure 3 | Completion of prelamin A maturation into lamin A depends on threestep

myeloproliferation phenotypes induced by concurrent

in a variety of cancer cell lines invitro and in multiple

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Box 3 | Protein prenylation in parasite biology and viral assembly

hypomorphic allele of Icmt into the Zmpste24 / mouse

Conclusions and perspectives

10 | ADVANCE ONLINE PUBLICATION

23. Mumby,S.M., Casey,P.J., Gilman,A.G., Gutowski,S.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

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12 | ADVANCE ONLINE PUBLICATION

109. Michaelson,D. etal. Postprenylation CAAX processing

nonfarnesylated version of progerin. Hum. Mol.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

165. Mammen,A.L. & Amato,A.A. Statin myopathy:

The authors thank the reviewers for helpful comments on the

Competing interests statement

The authors declare no competing interests.

ADVANCE ONLINE PUBLICATION | 13

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