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首页> 外文期刊>Biochemistry >Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependentand independent of phosphorylation of activation loop (T410) acidautophosphorylation (T560) sites
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Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependentand independent of phosphorylation of activation loop (T410) acidautophosphorylation (T560) sites

机译：胰岛素和PIP3通过既依赖又独立于激活环（T410）酸自磷酸化（T560）位点的磷酸化的机制激活PKC-zeta

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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.
机译：胰岛素激活蛋白激酶C-zeta（PKC-zeta）需要磷脂酰肌醇（PI）3-激酶依赖的磷脂酰肌醇-3,4,5-（PO4）（3）（PIP3）的增加以及激活环的磷酸化和自磷酸化网站，但实际机制尚不确定。目前，我们检查了：（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）突变的影响，以及（ii ）胰岛素诱导的PKC-zeta活化的N末端截短。胰岛素会急剧增加T410环的磷酸化作用，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-zeta和T410E-PKC-zeta的体外自磷酸化增加，但是胰岛素和PIP3并未刺激PKC-zeta的T560A，T560E，T410A / T560E，T410E / T560A或T410E / T560E突变体的自磷酸化。因此，T560似乎是唯一的自磷酸化位点。胰岛素和/或PIP3对酶活性的激活作用在T410A-PKC-zeta中被完全消除，在T560A-PKC-zeta，T410E / T560A-PKC-zeta和T410A / T560E-PKC-zeta中部分受损。在T410E-PKC-zeta，T560E-PKC-zeta和T410E / T560E-PKC-zeta中。 T410E / T560E突变体的激活提示磷酸化独立机制。作为功​​能相关性，T410A-PKC-zeta，T560A-PKC-zeta，T410E / T560A和T410A / T560E-PKC-zeta的表达损害了胰岛素对表位标记的GLUT4易位的影响，但T410E-PKC-zeta，T560E却没有-PKC-zeta或T410E / T560E-PKC-zeta。胰岛素而不是PIP3通过渥曼青霉素敏感的机制激活了PKC-zeta和PKC-lambda的截短的，缺乏伪底物的形式，这显然涉及PI 3-激酶/ PDK-1依赖性磷酸化，但不依赖于PIP3依赖性构象活化。研究结果表明胰岛素通过PIP3通过（a）PDK-1依赖性T410环磷酸化，（b）T560自磷酸化和（c）磷酸化非依赖性/构象依赖性的伪底物释放而引起PKC-zeta酶活性增加自抑制。

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