Chapter 19.
PROTEIN KINASE C
Overview of Insulin-sensitive
Signaling System
Insulin-sensitive Phospholipid
Signaling Pathways
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Major Phospholipid effect of insulin:
 GPI and PC hydrolysis in membrane
 de novo synthesis of PA in ER
 synthesis of PI in ER
 activation of PI3K in mambrane and ER
Signaling substances: IPG, DAG, PI3,4,5P3
DAG from 3 source can activate conventional and novel
PKCs : PC hydrolysis account for most of the initial burst of
DAG/PKC signaling in membrane
Insulin-sensitive hydrolytic and synthetic phospholipid
pathways are interrelated and integrated.
Via Rho,ARF
Pertussis toxin-sensitive Gi protein
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Phospholipid pathway are adapted to
provide for
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Rapid hydrolysis and resynthesis of GPI and
PC
Generation of phospholipid-derived signal
substance: IPG mediators, DAG,
polyphosphoinositides, PI3,4,5P3, PI3,4P2
Effects of Insulin on PC Hydrolysis
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Hydrolysis of PC DAG+phosphorylcholine or
PA+choline
DAG activate PKCs
Insulin-induced PC↓: very rapid, short-lived
 rapid resynthesis through the de novo pathway
IR/PC-PLD (?) : dependent PI3K
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small G protein, Rho and ARF can activate PC-PLD
Insulin via PI3K translocate or activate both Rho, ARF,
GRP1(ARF exchange factor)
Effects of Insulin On PI 3-kinase
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↑D3 PO4 polyphosphoinositides(PI3,4,5P3/PI3,4P2)
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Activate PDK1, PKB, aPKCs, DAG-dep PKCs, PRK1/2/3
Mambrane localizing factor to bind and coordinate
signaling factors
Translocate small G proteins,Rho ARF
Activate Rac, Rab for cell ruffling and GLUT-4
translocation
By Tyrosin phospholylation of IRS family
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pYXXM of IRS / SH2 on p85 of PI3K
Effects of Insulin On DAG
Production
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Insulin provokes rapid increase in DAG
mass
in DAG production
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Initial burst : PC hydrolysis in memb
Later: de novo PA synthesis in ER
General Aspects of PKC Activation
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5 types
 conventional or classic cPKCs(,1, 2,)
 Novel nPKCs(,,,)
 Atypical aPKC(,,)
 Membrane-anchored PKCs(PKC-m or PKD)
 PRK 1,2,3
Activated by DAG, Ca++, PI3,4,5P3, PA
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DAG, Polyphosphoinositides, lipids activate PKCs
Full activation of all PKCs: phosphorylation of Thr-,
Ser- in “activation loop”/autoP in catalytic domains
binding of DAG or PI3,4,5P3 molecular unfolding at
V3allosteric dissociation of V1 autoinhibitory
pseudosubstratephosphorylated/activated
vulnable to proteasecleavage and release of
regulatory/catalytic frag.
Activation of all PKCs requires phospholylation of
specific sites in activation loops by PDK1:
cPKCs,nPKCs vs aPKCs
PI3,4,5P3 activate PDK1 directly or unfold aPKCs to
expose the loop site to PDK1
Effects of Insulin On Activation of
DAG-dependent PKC isoforms
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Insulin-induced DAGPKC activation? Membrane
PKC enzyme activity? : cPKCs/nPKCs
Activation of cPKCs/nPKCsPC hydrolysis or de
novo PA synthesis? : wartmannin(PI3K inhibitor)
Insulin strongly activate aPKCs: Activation of PKC
may have been primarily reflective of activation of
aPKCs rather than, or as well as, cPKC/nPKCs
PKC downregulation
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The biological effect of Insulin do not require
DAG/PKC signaling?
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Acute phorbol ester-induced PKC depletion effects of
insulin persist
PKC-,- : relatively resistant to downregulation
The biological effect of Insulin require DAG/PKC
signaling?
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Chronic phorbol ester Tx effects of insulin
Nonspecific? Activation of residual PKCs?
Downregulation of insulin signaling mechanisms?
Insulin-like effects of Phorbol esters
Gluc transport↑
 Activation of enz in intracelluar gluc metabolism
 Activation of acetyl-CoA carboxylase
 Ion transport enz ↑: Na+/H+ transporter,
Na+/K+ATPase
 AA transport↑
 Activation of protein synthesis initiation and
elongation factors
 Change in gene expression
 DNA synthesis ↑ in some cell type
Insulin effect: DAG-dependent PKCs?
Phorbol ester may activate PI3K, Raf, ERK
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PKC Inhibitors
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Inhibitors can be helpful in determining which
metabolic processes are likely or not likely to
be regulated by PKC
 Inhibit cPKCs at low conc./nPKCs at
intermediate conc./aPKCs at high conc.
 Not entirely specific for PKCs
 Phorbol ester + inhibitor
 GO6976 : Selectively inhibit PKC ,,
The role of DAG-sensitive PKC in
Glucose Transport ?
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Effect of Phorbol ester on glu transport are much less than
insulin
DAG itself increase glu transport ; PLC-induced,
electrical/exercise-induced DAG
DAG-dep PKCs are not required for activating gluc transport in
rat adipocyte, myotube, myocytes
PKC-, PKC- knockout mice : 오히려 insulin effect 증가
 tonic inhibitory effect of cPKCs on activation of PI3K/aPKCs
Phorbol ester-induced PKC downregulation of all cPKC(,):
insulin effects on GLUT-4 translocation/glu transport 유지.
Inhibitor study : selective inhibitor of cPKCsno effect
: aPKCs rather than cPKCs or nPKCs may be required for
glucose transport effect on insulin
Glucose-induced Activation of
DAG-sensitive PKC
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Extracellular Glu: de novo PA synthesis↑, activate DAGsensitive PKCs  diabetic Cx/ IR?
Adipocyte vs skeletal m. with persistent
hyperglycemia,hyperinsulinemia
DAG-sensitive PKCsGlucose-induced IR in tissue?
 DAG-dep PKCs may impair insulin-induced activation IR
tyrosine kinase ?
 DAG-dep PKCs may inhibit IRS-dep PI3K activation by
activation of MAP kinase ?
 Acute phorbol ester-induced PKC activationno effect
 DAG/PKC in IR, but normal tyrosin kinase/PI3K activation
The role of Atypical PKCs in Insulin Action
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Activation of PKCζ and PKCλ : phosphorylation of PDK-1dep
activation loop sites auto- or trans-phosphorylation
By PI3K
 Inhibited by PI3K inhibitor or DN p85 subunits of PI3K
 Activated by phosphotyrosine-containing peptide(pYXXM)
activators of PI3K and by direct addition of PI3,4,5P and
PI3,4P2
By PDK1(phosphorylate Thr-410, Thr-411)
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Overexpression of WT PDK1/kinase-inactive PDK1/activation
resistant (Thr-410Ala) mutant PKCζ
PI3,4,5P3 may act
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by directly activating PDK1
by interacting with PKCs to facilitate interaction with PDK1
by increasing autophophorylation and allosterically relieving
pseudosubstrate-dependent autoinhibition
The role of PKCζ and PKCλ in Insulinstimulated Glucose Transport
Stable Transfection study
: inactive PKCζ GLUT 4 translocation, glu uptake 
: wild type PKCζ GLUT4 translocation, glu uptake 
 Transient transfection and gene transfer study
 Insulin stimulation of HA-GLUT-4 translocation is inhibited
by kinase-inactive form of PKCζ and PKCλ mutant :
rescued interchangeably by wild-type of either PKCζ or
PKCλ
 Kinase-inactive PDK1, mutant PKCζ
 Adenoviral gene transfer study
 PKCλ knock-out cell
 aPKCs and putative upstream activators are required for
insulin-stimulated Glucose Transport in adipocyte and
myocytes
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Upstream
 aPKCs operate along with other signaling factors in
specific intracellular sites : Cbl and Rho family
member(TC10)
 Cbl-dep PI3K, IRS-1-dep PI3K
downstream
 aPKCs phophorylate and regulate the SNARE protein
VAMP2(translocation of GLUT-4 from ER to memb)
 Phosphorylation of insulin-responsive
aminopeptidase(IRAP)(promote GLUT-4 translocation)
Defects in Activation of PKCζ and
PKCλ in Obesity,T2DM, and Other
insulin-resistant states
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Defect in aPKC activation by insulin in muscle of
obese, glucose intolerant, and T2DM humans and
monkeys
 Defective activation of upstream regulators: IRS1/2
 Defective responsiveness of aPKCs to PI3,4,5P3
The role of PKCζ and PKCλ in ERK
Activation by Insulin
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In some cells, inhibitors of PI3K inhibit insulininduced ERK ½
Transient cotransfection study
: kinase inactive PKCζ/PDK1, mutant PKCζ
inhibit ERK activation
Additional requirement of Grb2, SOS, Ras, Raf etc.
: aPKC are needed to activate Raf
The role of PKCζ and PKCλ in
Activation of Protein Synthesis
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Initiation of Protein systhesis by insulin
 by phosphorylation-dependent inhibition of PHAS1/2(constitutively inhibit initiation factor eIF-4E)
 Direct phosphorylation of eIF-4E
 Activation of p70 S6 kinase
PI3K,PKB for PHAS, but aPKCs?
 PDK1 for phosphorylation of Thr-252 in p70 S6 kinase
 aPKCs for phosphorylation of Thr-412 in p70 S6 kinase
full activation of p70 S6 kinase?
Role of aPKCs in gene expression of insulin?
The role of Atypical PKC in PI3Kindependent Activation of Glucose
Transport by Noninsulin Agonists
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High glu/sorbitol activate PYK2 via osmotic sensor
AICAR, DNP activate PYK2 via AMPK
PYK2Grb2/SOS/Ras/Raf/MEK1/ERK pathway PLD
activation
PLD activation PAaPKC activationtranslocation
GLUT4, glu uptake
Exercise activate aPKCs & ERK via AMPK
TZDs activate aPKCs via Cbl-dep PI3K
increase IRS1/2 (Adipocyte: /Skeletal m : )
Conclusion