Chapter 19. PROTEIN KINASE C

Overview of Insulin-sensitive Signaling System

Insulin-sensitive Phospholipid Signaling Pathways

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,ARFPertussis toxin-sensitive Gi protein

Phospholipid pathway are adapted to provide for Rapid hydrolysis and resynthesis of GPI and P

C Generation of phospholipid-derived signal subst

ance: IPG mediators, DAG, polyphosphoinositides, PI3,4,5P3, PI3,4P2

Effects of Insulin on PC Hydrolysis

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

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

↑D3 PO4 polyphosphoinositides(PI3,4,5P3/PI3,4P2) Activate PDK1, PKB, aPKCs, DAG-dep PKCs, PRK1/2/3 Mambrane localizing factor to bind and coordinate signali

ng factors Translocate small G proteins,Rho ARF Activate Rac, Rab for cell ruffling and GLUT-4 translocati

on By Tyrosin phospholylation of IRS family

pYXXM of IRS / SH2 on p85 of PI3K

Effects of Insulin On DAG Production

Insulin provokes rapid increase in DAG mass

in DAG production Initial burst : PC hydrolysis in memb Later: de novo PA synthesis in ER

General Aspects of PKC Activation

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

DAG, Polyphosphoinositides, lipids activate PKCs Full activation of all PKCs: phosphorylation of Thr-, S

er- in “activation loop”/autoP in catalytic domains binding of DAG or PI3,4,5P3 molecular unfolding at

V3allosteric dissociation of V1 autoinhibitory pseudosubstratephosphorylated/activated vulnable to proteasecleavage 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

Insulin-induced DAGPKC activation? Membrane PKC enzyme activity? : cPKCs/nPKCs

Activation of cPKCs/nPKCsPC 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

The biological effect of Insulin do not require DAG/PKC signaling? Acute phorbol ester-induced PKC depletion effects of insuli

n persist PKC-,- : relatively resistant to downregulation

The biological effect of Insulin require DAG/PKC signaling? 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 fa

ctors Change in gene expression DNA synthesis ↑ in some cell type Insulin effect: DAG-dependent PKCs? Phorbol ester may activate PI3K, Raf, ERK

PKC Inhibitors 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 interm

ediate 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 ?

Effect of Phorbol ester on glu transport are much less than insulin DAG itself increase glu transport ; PLC-induced, electrical/exercise-indu

ced DAG DAG-dep PKCs are not required for activating gluc transport in rat adip

ocyte, 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 effe

cts on GLUT-4 translocation/glu transport 유지 . Inhibitor study : selective inhibitor of cPKCsno effect : aPKCs rather than cPKCs or nPKCs may be required for glucose transport effect on insulin

Glucose-induced Activation of DAG-sensitive PKC

Extracellular Glu: de novo PA synthesis↑, activate DAG-sensitive PKCs diabetic Cx/ IR?

Adipocyte vs skeletal m. with persistent hyperglycemia,hyperinsulinemia

DAG-sensitive PKCsGlucose-induced IR in tissue? DAG-dep PKCs may impair insulin-induced activation IR tyr

osine kinase ? DAG-dep PKCs may inhibit IRS-dep PI3K activation by acti

vation of MAP kinase ? Acute phorbol ester-induced PKC activationno effect DAG/PKC in IR, but normal tyrosin kinase/PI3K activation

The role of Atypical PKCs in Insulin Action

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) Overexpression of WT PDK1/kinase-inactive PDK1/activation resista

nt (Thr-410Ala) mutant PKCζ PI3,4,5P3 may act

by directly activating PDK1 by interacting with PKCs to facilitate interaction with PDK1 by increasing autophophorylation and allosterically relieving pseudos

ubstrate-dependent autoinhibition

The role of PKCζ and PKCλ in Insulin-stimulated 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 insuli

n-stimulated Glucose Transport in adipocyte and myocytes

Upstream aPKCs operate along with other signaling factors in specifi

c intracellular sites : Cbl and Rho family member(TC10) Cbl-dep PI3K, IRS-1-dep PI3K

downstream aPKCs phophorylate and regulate the SNARE protein VAM

P2(translocation of GLUT-4 from ER to memb) Phosphorylation of insulin-responsive aminopeptidase(IRA

P)(promote GLUT-4 translocation)

Defects in Activation of PKCζ and PKCλ in Obesity,T2DM, and Other ins

ulin-resistant states Defect in aPKC activation by insulin in muscle of ob

ese, glucose intolerant, and T2DM humans and monkeys Defective activation of upstream regulators: IRS-1

/2 Defective responsiveness of aPKCs to PI3,4,5P3

The role of PKCζ and PKCλ in ERK

Activation by Insulin In some cells, inhibitors of PI3K inhibit insulin-indu

ced 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

Initiation of Protein systhesis by insulin by phosphorylation-dependent inhibition of PHAS-1/2(con

stitutively 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 PI3K-independent Activation of Glucose Transport by Non

insulin Agonists High glu/sorbitol activate PYK2 via osmotic sensor AICAR, DNP activate PYK2 via AMPK PYK2Grb2/SOS/Ras/Raf/MEK1/ERK pathway PLD acti

vation PLD activation PAaPKC activationtranslocation GLU

T4, glu uptake Exercise activate aPKCs & ERK via AMPK TZDs activate aPKCs via Cbl-dep PI3K

increase IRS1/2 (Adipocyte: /Skeletal m : )

Conclusion