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Presentation on theme: "Chapter 19. PROTEIN KINASE C"— Presentation transcript:

1 Chapter 19. PROTEIN KINASE C

2 Overview of Insulin-sensitive Signaling System



5 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.

6 Pertussis toxin-sensitive Gi protein
Via Rho,ARF Pertussis toxin-sensitive Gi protein

7 Phospholipid pathway are adapted to provide for
Rapid hydrolysis and resynthesis of GPI and PC Generation of phospholipid-derived signal substance: IPG mediators, DAG, polyphosphoinositides, PI3,4,5P3, PI3,4P2

8 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)

9 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 signaling factors Translocate small G proteins,Rho ARF Activate Rac, Rab for cell ruffling and GLUT-4 translocation By Tyrosin phospholylation of IRS family pYXXM of IRS / SH2 on p85 of PI3K

10 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

11 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


13 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

14 Effects of Insulin On Activation of DAG-dependent PKC isoforms
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

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

16 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

17 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 intermediate conc./aPKCs at high conc. Not entirely specific for PKCs Phorbol ester + inhibitor GO6976 : Selectively inhibit PKC ,,

18 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-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

19 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 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

20 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 resistant (Thr-410Ala) 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 pseudosubstrate-dependent autoinhibition

21 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 insulin-stimulated Glucose Transport in adipocyte and myocytes

22 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)

23 Defects in Activation of PKCζ and PKCλ in Obesity,T2DM, and Other insulin-resistant states
Defect in aPKC activation by insulin in muscle of obese, glucose intolerant, and T2DM humans and monkeys Defective activation of upstream regulators: IRS-1/2 Defective responsiveness of aPKCs to PI3,4,5P3

24 The role of PKCζ and PKCλ in ERK Activation by Insulin
In some cells, inhibitors of PI3K inhibit insulin-induced 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

25 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(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?

26 The role of Atypical PKC in PI3K-independent Activation of Glucose Transport by Noninsulin Agonists
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 : )

27 Conclusion



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