K325 CELLBIOLOGYLABORATORY

SIGNALTRANSDUCTIONANDPROTEINKINASEA
Perceptionof manyextracellular
signalsleadsto productionof second
messengers within the responding
cells. Secondmessengers are small
molecules or ions. Important
examplesof secondmessengersare:
;1(' figure 615 TheSe,ondMessenge,Con,ept Various hor-
cyclicAMP,Ca
2
+, inositol
mones, neurotransmitters,andlocal mediators function as
first messengersthatbindto theplasmamembraneand trisphosphate(IP3)'anddiacylglycerol.
trigger theformation ofother signaling molecules, called
second messengers, whichfunctioninsidethe cell,
Amplification
Amplification
Epinephrine
t ~
t:,. t:,. t:,.
t ~ ~

t t t t t
Adenylate
cyclase
cAMP 10-
6
M
Duringsignal transduction,
signal amplificationoccurs. In
additionto secondmessengers,
signaltransductionpathways
utilize the activation (or
deactivation) of particular
enzymes.
o 0 0 0 0 Kinase
Amplification
Amplification
t ~ ~
CD ~ $ \it <i)
t ~ ~

Activated
enzyme
Product
+ ... Figure 1924 Cellular transduction and amplification of
~ ~ an extracellular signal. In this example, binding a single epi-
nephrine molecule results in the synthesis of a large number
of cAMP molecules, which, in turn, activate multiple enzyme
molecules.
There are varietyof mechanisms for
regulatingenzyme activity. A
prominent mechanism in signal
transductionis a typeof covalent
modification called phosphorylation.
<8>=phosphate. methyl. acetyl,
ADP-ribosyl, etc.
I
*figure 222 M.,han;smsEmployedby Cells to Regulate
Enzyme Ad;v;ty Thebindingof allosteric regulators, the
covalentmodificationofproteinstructure. andthe associa-
tionanddissociationofproteinsubunitscan beemployed
". ~ +
~
eitherto increase ordecrease the catalyticactivityof an
enzymemolecule.
IL----
1
_
-------- ---------
o
Protein kin.oe I ADP +

I
0-
o
Protem 0
I II pllOSplwlo5C IP '"t 'I 'f-o II
H20 + Prote.rL 0 -i-O- I '(0 en H + HO-P-O-
0- H20 6-
+
.. Figure 1919 Reactions catalyzed by protein kinase and
protein phosphatase. The phosphorylated and dephosphoryl-
ated forms of the protein often differ markedly in enzymatic
reactivity,
+
PhosPhat:<
'!/ p
Two dimers of One tetramer of 4<ff>
phosphorylase b phosphorylase a
(inactive) (active)
Figure 223 Contol 01 Glycogen Phosphorylase Activity by
Phosphorylation and Dephosphorylation Thephosphory-
lation of each subunit and the resulting joining of two
dimers to form a tetramer results in enzy'ne activation.

HO OH
1 M'""" cyclase
ICyclic AMPI
NH
2
;:J"
N

HO OH
>t Figure 614 The Formation and Breakdown 01 Cyt/it
AMI' Adenylyl cyclase, an enzyme located in the plasma
11....-....-
membrane, catalyzes the formation of cyclic AMPfrom ATP.
CyclicAMP is broken down to AMP by the cytoplasmic
enzyme, phosphodiesterase.
2
Protein kinases phosphorylate
proteins whereas protein
phosphatases dephosphorylate
proteins. Protein kinases are major
components of signal transduction
pathways. Phosphorylationof the
substrate protein alters its activity
(either activation or deactivation).
Glycogen phosphorylase is a classic
example of an enzyme that is activated
by phosphorylationand deactived by
dephosphorylation. The balance
between kinase and phosphatase
activity has a major Impact on the
activation state of the substrate
enzyme.
Cyclic AMP is produced by adenylyl
cy1case and degraded by cAMP
phosphodiesterase.
-
Receptor G. protein
Activation of a receptor activates a
trimeric G protein. In turn, this
activated G protein activates adenylyl
cyclase.
Signaling
m o l u l ~
Table 64 Some Hormonal Responses Associated
>K with Increased Cyclic AMP Levels
Hormone Tisslie Response
ACTH Adrenal Hydrocortisone formation
TSH Thyroid Thyroxine formation
ill Ovary Progesterone formation
Vasopressin Kidney Water resorption
Glucagon Adipose tissue lipid breakdown
Glucagon Liver Glycogen breakdown
Parathormone Kidney Phosphate excretion
Parathormone Bone Bone resorption
Secretin Intestine Pancreatic enzyme release
Epinephrine Cardiac muscle Increased contractility
Epinephrine liver Glycogen breakdown
Epinephrine Erythrocyte Increased Na+ permeability
3
Figure 617 Mec"anism by W"ic" f"e G, Profein Mediafes
f"e Inferadion befween a Recepfor and Adenylyl
Cyclase The binding of certain signaling molecules to their
plasma membrane receptors causes the receptor to bind to
G, (a ttpta-ancboreaplasma membrane protein). This Inter-
action triggers the dissociation of GDPfrom the G, protein
and its replacement by GTP. In the presence of bound GTP,
the a subunit dissociates from the G, protein and activates
adenytyi cyclase. When GTP is subsequently hydrolyzed, the
a subunit reassociates with f3 and y subunits and the ability
to stimulate adenytyt cyclase Is lost.
In vertebrates, numerous hormones
elicit responses in the appropriate
tissues Via increases in cAMP levels.
I
Increases in cAMP concentrations
activate protein kinase A (PKA).
Regulatory
subunit
Catalytic
subunit
Protein kinase A
(inactive) Protein
kinase A
(active)
Figure 6-19 Adilfation of ProteinKinase Aby Cyclic
AMP CyclicAMP causes the regulatory subunits ofprotein
kinase A to dissociate from the catalytic subunits; the free
catalytic subunits in turn catalyze protein phosphorylation.
Phosphorylase kinase
Glycogen synthase
Protein phosphatase-I
Protein phosphatase inhlbitor-I
,Hot'Il1()ne-sensitive lipase
Pyruvate kinase: .'"
6-Phosphofructo-Z-kinase
." , ',':;
Tyrosine'hydroxylase ....
.. ..
Phenylalanine hydroxylase
Troponin I
PrAdrenergic receptor
Tubulin
Cyclic AMPresponse element binding protein (CREB)
'If" Epinephrine
t
There are several known examples of
proteins whose activities are regulated
by being phosphorylated by PKA
The cell-type specificity of cAMP
responses IS caused in part by the celI-
type specificity of the PKA substrates.
LIVER CELL FAT CELL HEART MUSCLE
CELL
% Figure 622 Mechanism by Which Hormones Using 'he
SameSecond Messenger(Cyclic AMP] Elicit Different
Responses in DifferentCellTypes Protein kinase A activ-
ity is elevated in liver andfat cells stimulated by glucagon,
and in heart muscle cells stimulated by epinephrine. Yet the
effects of the elevated protein kinase A activity are dffJerent
in the three cell types because each cell contains dffJerent
proteins that are phosphorylated by protein kinase A.
4
IL.-- _
The breakdownof glycogeninliveris a classicexampleof a hormonalresponse
mediatedbycAMPandPKA.
Adenylyl
cyclase
Plasma
membrane
.
Acilvates ( Phosphorylase kinase
I

@]Glycogen
Inactivates
'--___+( Protein phosphatase inhibitor-1
Protein phosphatase inhibitor-1 (inhibits enzyme [I] )
Figure621 Pathwayby Whi,h Gly(ogen BreakdownIsStimulatedby Hormones ThatElevateCydi(AMPLevels CycliC
AMPactivatesproteinkinaseA,which catalyzesthephosphorylationofphosphorylasekinase(becomes activated),glycogen
synthase(becomes Inactivated), proteinphospbatase-I(becomes Inactivated), andproteinphosphataseinbibitor-I (becomes
activated).Thesechanges in enzymeactivitylead toan enhancementofglycogen breakdowncatalyzedbyglycogenphospho-
rylase, andan inhibitionofglycogensynthesis catalyzedbyglycogen synthase.
Someenzymesare deactivatedbyincreasingcAMPlevels. Aclassic exampleis
phosphoproteinphosrhatase1(PPl). After arise incAMP,PKA phosphorylatesI-I
(aproteininhibitor0 PPl),whichthenbindsto andinactivatesPPI.
Protein inhibitor
(inactive)
cAMP-dependent
protein kinase
7 "'\I)

.. 0 . . P
a e
AlP ADP
I
+
... Figure 1923 Inhibition of phosphoprotein phosphatase
protein kinase to bind to and inhibit'the phosphoprotein
bycAMP.Phosphoprotein phosphatase isenzymaticallyac-
phosphatase. Thus the phosphatase isinactive in the presence
tive,exceptwhen an inhibitor protein isbound to it. The
of a high levelof cAMP and activeonly when the levelof
inhibitor must be phosphorylated bythe cAMP-dependent
cAMPislow. [SeeP.Cohen, 1982, Nature 296:613.)
5
Adrenalln

PAdrenergic Gs
receptor
r
Figure 2. CREB Pathway
Aschematic illustration of the PKs and PPs that regulate activation
of the CREB transcription factor.
1<-
Target GProtein lctlon
Adenylyl cyclase G, ,Stimulatesenzyme
G, Inhibits enzyme
K+ channel G, Opensionchannel
PhospholipaseC c,
CyclicGMP G,(transducin) Stimulatesenzyme
phosphodiesterase'
1"------
'Therole ofthe G,proteininregulating phosphodiesteraseis
disCussedinChapter17.
Some cAMP responses
involve the induction of
genes. A cAMP-responsive
genecontainsa regulatory
DNAsequence,caIledeRE (for
"AMPresponse
withinits promoter. A
transcriptionfactor called
CREB (forCRE binding
protein)isactivatedbyPKA.
PhosphorylatedCREB bindsto
theeRE, resultingin
transcriptionof the gene.
There are multiple trimeric G proteins
andmultiple targets.
6
_
Another major signal transduction pathway involves the G protein and
phospholipase C. Phospholipase C produces diacylglycerol and IP3' IP3 causes
cytosolic Ca
2
+ to rise. The nse in DA.G and Ca
2
+ activates protein kinase C.
11
3
a'
Ca
2
released
from endoplasmic
reticulum
Figure 623 The Phosphoinositide Pathway for Producing the Second Messengers Inositol Trisphosphate (IP3) and
Diacylglycerol (DAG) In this pathway, binding of a signaling molecule to its receptor activates a G
p
protein, which in turn
activates phospholipase C. Phospholipase C, which normally resides In the cytosol, is recruited to the inner surface of the
plasma membrane, where it catalyzes the formation ofDAG and IP
3.
DAGfunctions by activating protein kinase C, while IP]
mobilizes the release of Ca
2
from the endoplasmic reticulum.
A variety of stimuli activate
phospholipase C.
Signaling
molecule
\

" . ,"00"'0_ C h c ,
Molecule Target Cell Response
Epinephrine Liver (ai-receptor) Glycogen
breakdown
Vasopressin Liver , Glycogen
.
breakdown
PDGF Fibroblasts Cell proliferation
Acetylcholine Smooth muscle Contraction
(muscarinic receptor)
Structure of the Lissamine Rhodamine B - Kemptide substrate for PKA.
I
Leu-Arg-Arg-Ala-Ser-Leu-Gly
7
1---------------------
, . Enzymeconcentration = 2x
[5]
When substrate concentrations are in
excess, the amount of product formed
in an enzyme reaction is directly
proportional to the concentration of
enzyme in the reaction.
Figure 27 Effect of EnzymeContenlralion on Ille Role of
an EnzymeCaialyzedReaction (fop) In a plot of reaction
velocity versus substrate concentration, tbe reaction Is seen
to proceed at balf Its maximal velocity uiben tbe substrate
concentration Is equal to tbe Micbaetis constant (K",). When
the enzyme concentration Is doubled, tbe maximal velocity
(V ....,J Is also doubled, but K
m
stays tbe same. (Bottom) In a
Llneweaver-Burk plot of the same data, tbe y Intercept corre-
sponds to tbe reciprocal ofV.. and the x Intercept and
\
,,---.
equals tbe negative reciprocal of Km
Figures from:
* Kleinsmith, L.J. and Kish, V.M. (1995) Principles of Cell and Molecular Biology
(2nd edition). HarperCollins College Publishers, New York.
+ Darnell, J., Lodish, H. and Baltimore, D. (1990) Molecular Cell Biology (2nd
edition). Scientific American Books, New York.
t Hunter, T. (1994) Protein kinases and phosphatases. The yin and yang of protein
phosphorylation and signaling. Cell 80: 225-236.
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I