胰岛素和磷脂酰肌醇-3,4,5-三磷酸（PIP3）通过依赖和不依赖激活环（T410）磷酸化及自身磷酸化（T560）位点的机制激活蛋白激酶C-ζ（PKC-ζ）。
Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites.
作者信息
Standaert M L, Bandyopadhyay G, Kanoh Y, Sajan M P, Farese R V
机构信息
J. A. Haley Veterans' Hospital Research Service, and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
出版信息
Biochemistry. 2001 Jan 9;40(1):249-55. doi: 10.1021/bi0018234.
Activation of protein kinase C-zeta (PKC-zeta) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO(4))(3) (PIP(3)) 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-1 (PDK-1). Despite increasing in vitro autophosphorylation of wild-type PKC-zeta and T410E-PKC-zeta, insulin and PIP(3) 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 PIP(3) 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 PIP(3), 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 PIP(3)-dependent conformational activation. Our findings suggest that insulin, via PIP(3), 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.
胰岛素对蛋白激酶C-ζ（PKC-ζ）的激活需要磷脂酰肌醇（PI）3-激酶依赖性地增加磷脂酰肌醇-3,4,5-（PO(4)）(3)（PIP(3)）以及激活环的磷酸化和自磷酸化位点，但实际机制尚不确定。目前，我们研究了：（a）胰岛素对苏氨酸（T）-410环磷酸化的急性影响，以及（b）（i）T410环和T560自磷酸化位点的丙氨酸（A）和谷氨酸（E）突变，以及（ii）N端截短对胰岛素诱导的PKC-ζ激活的影响。胰岛素急性增加T410环磷酸化，提示3-磷酸肌醇依赖性蛋白激酶-1（PDK-1）的作用增强。尽管野生型PKC-ζ和T410E-PKC-ζ的体外自磷酸化增加，但胰岛素和PIP(3)并未刺激T560A、T560E、T – 410A/T560E、T410E/T560A或T410E/T560E突变形式的PKC-ζ的自磷酸化；因此，T560似乎是唯一的自磷酸化位点。胰岛素和/或PIP(3)对酶活性的激活作用在T410A-PKC-ζ中完全消除，在T560A-PKC-ζ、T410E/T560A-PKC-ζ和T410A/T560E-PKC-ζ中部分受损，而在T410E-PKC-ζ中的T560E-PKC-ζ和T410E/T560E-PKC-ζ中基本保持完整。T410E/T560E突变体的激活提示了一种不依赖磷酸化的机制。作为功能相关性，胰岛素对表位标记的GLUT4易位的影响因T410A-PKC-ζ、T560A-PKC-ζ、T410E/T560A和T410A/T560E-PKC-ζ的表达而受损，但不受T410E-PKC-ζ、T560E-PKC-ζ或T410E/T560E-PKC-ζ的影响。胰岛素而非PIP(3)通过渥曼青霉素敏感机制激活截短的、缺乏假底物的PKC-ζ和PKC-λ形式，显然涉及PI 3-激酶/PDK-1依赖性磷酸化，但独立于PIP(3)依赖性构象激活。我们的研究结果表明，胰岛素通过PIP(3)，通过（a）PDK-1依赖性T410环磷酸化、（b）T560自磷酸化和（c）不依赖磷酸化/依赖构象的假底物自抑制解除，引起PKC-ζ酶活性增加。
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