Insulin and PIP3 activate PKC-ζ by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) acid autophosphorylation (T560) sites

被引:114
作者
Standaert, ML
Bandyopadhyay, G
Kanoh, Y
Sajan, MP
Farese, RV
机构
[1] James A Haley Vet Hosp, Res Serv, Tampa, FL 33612 USA
[2] Univ S Florida, Coll Med, Dept Internal Med, Tampa, FL 33612 USA
关键词
D O I
10.1021/bi0018234
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Activation of protein kinase C-zeta (PKC-zeta) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO4)(3) (PIP3) and phosphorylation of activation loop and autophosphorylation sites, but actual mechanisms are uncertain. Presently, we examined: (a) acute effects of insulin on threonine (T)-410 loop phosphorylation and (b) effects of (i) alanine (A) and glutamate (E) mutations at T410 loop and T560 autophosphorylation sites and (ii) N-terminal truncation on insulin-induced activation of PKC-zeta. Insulin acutely increased T410 loop phosphorylation, suggesting enhanced action of 3-phosphoinositide-dependent protein kinase-l (PDK-1). Despite increasing in vitro autophosphorylation of wild-type PKC-zeta and T410E-PKC-zeta, insulin and PIP3 did not stimulate autophosphorylation of T560A, T560E, T410A/T560E, T410E/T560A, or T410E/T560E mutant forms of PKC-zeta; thus, T560 appeared to be the sole autophosphorylation site. Activating effects of insulin and/or PIP3 on enzyme activity were completely abolished in T410A-PKC-zeta, partially compromised in T560A-PKC-zeta, T410E/T560A-PKC-zeta, and T410A/T560E-PKC-zeta, and largely intact in T410E-PKC-zeta, T560E-PKC-zeta, and T410E/T560E-PKC-zeta. Activation of the T410E/T560E mutant suggested a phosphorylation-independent mechanism. As functional correlates, insulin effects on epitope-tagged GLUT4 translocation were compromised by expression of T410A-PKC-zeta, T560A-PKC-zeta, T410E/T560A, and T410A/T560E-PKC-zeta but not T410E-PKC-zeta, T560E-PKC-zeta, or T410E/T560E-PKC-zeta. Insulin, but not PIP3, activated truncated, pseudosubstrate-lacking forms of PKC-zeta and PKC-lambda by a wortmannin-sensitive mechanism, apparently involving PI 3-kinase/PDK-1-dependent phosphorylations but independent of PIP3-dependent conformational activation. Out findings suggest that insulin, via PIP3, provokes increases in PKC-zeta enzyme activity through (a) PDK-1-dependent T410 loop phosphorylation, (b) T560 autophosphorylation, and (c) phosphorylation-independent/conformational-dependent relief of pseudosubstrate autoinhibition.
引用
收藏
页码:249 / 255
页数:7
相关论文
共 16 条
[1]   Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B alpha [J].
Alessi, DR ;
James, SR ;
Downes, CP ;
Holmes, AB ;
Gaffney, PRJ ;
Reese, CB ;
Cohen, P .
CURRENT BIOLOGY, 1997, 7 (04) :261-269
[2]  
Bandyopadhyay G, 1997, J BIOL CHEM, V272, P2551
[3]   Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-ζ [J].
Bandyopadhyay, G ;
Standaert, ML ;
Sajan, MP ;
Karnitz, LM ;
Cong, L ;
Quon, MJ ;
Farese, RV .
MOLECULAR ENDOCRINOLOGY, 1999, 13 (10) :1766-1772
[4]   Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ε) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes:: specific interchangeable effects of protein kinases C-ζ and C-λ [J].
Bandyopadhyay, G ;
Standaert, ML ;
Kikkawa, U ;
Ono, Y ;
Moscat, J ;
Farese, RV .
BIOCHEMICAL JOURNAL, 1999, 337 :461-470
[5]   Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes [J].
Bandyopadhyay, G ;
Standaert, ML ;
Galloway, L ;
Moscat, J ;
Farese, RV .
ENDOCRINOLOGY, 1997, 138 (11) :4721-4731
[6]   Regulation of protein kinase C ζ by PI 3-kinase and PDK-1 [J].
Chou, MM ;
Hou, WM ;
Johnson, J ;
Graham, LK ;
Lee, MH ;
Chen, CS ;
Newton, AC ;
Schaffhausen, BS ;
Toker, A .
CURRENT BIOLOGY, 1998, 8 (19) :1069-1077
[7]   The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction [J].
Cohen, P .
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, 1999, 354 (1382) :485-495
[8]   Primary structure, tissue distribution, and expression of mouse phosphoinositide-dependent protein kinase-1, a protein kinase that phosphorylates and activates protein kinase Cζ [J].
Dong, LQ ;
Zhang, RB ;
Langlais, P ;
He, HL ;
Clark, M ;
Zhu, L ;
Liu, F .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (12) :8117-8122
[9]   Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol [J].
Dutil, EM ;
Newton, AC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (14) :10697-10701
[10]   Requirement of atypical protein kinase Cλ for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes [J].
Kotani, K ;
Ogawa, W ;
Matsumoto, M ;
Kitamura, T ;
Sakaue, H ;
Hino, Y ;
Miyake, K ;
Sano, W ;
Akimoto, K ;
Ohno, S ;
Kasuga, M .
MOLECULAR AND CELLULAR BIOLOGY, 1998, 18 (12) :6971-6982