Leventhal P S, Bertics P J
Department of Biomolecular Chemistry, University of Wisconsin-Madison 53706.
J Biol Chem. 1993 Jul 5;268(19):13906-13.
Protein substrates for protein kinase C (PKC) have phosphorylation domains that are typically rich in the basic amino acids arginine and lysine. However, arginine-rich proteins may interact with PKC differently than lysine-rich proteins, i.e. lysine-rich histone requires phospholipid and Ca2+ to be phosphorylated, whereas the arginine-rich protein, protamine, can bypass these effector requirements. We have studied the interaction of PKC with protamine, histone, poly-L-arginine, and poly-L-lysine to better understand the role of basic protein domains in PKC activation, effector dependence, and substrate specificity. Using a microtiter binding assay, PKC was found to bind tightly to protamine and poly-L-arginine, but not to histone or poly-L-lysine, in the absence of phospholipid, Ca2+, and MgATP. Furthermore, poly-L-arginine was much more potent than poly-L-lysine at inhibiting protamine phosphorylation; i.e. 1-2 nM poly-L-arginine was sufficient to cause 50% inhibition of protamine phosphorylation, whereas over 300 microM poly-L-lysine was needed to reach 50% inhibition. Autophosphorylation of PKC in the absence of activators was potently stimulated by protamine and poly-L-arginine, but not by histone or poly-L-lysine, suggesting selective stimulation of PKC by arginine-rich polypeptides. Double-reciprocal plots of protamine phosphorylation using either a mixture of isozymes (alpha/beta/gamma) or isolated PKC-beta were parabolic, and analysis of the kinetic data on velocity/[protamine] versus velocity plots indicated positive cooperativity with respect to protamine. These findings are consistent with those from autophosphorylation experiments in that PKC appears to be selectively stimulated by arginine-rich polypeptides. These results suggest that PKC can preferentially bind arginine-rich proteins in the absence of phospholipid and Ca2+. This interaction appears to be distal to the catalytic site and thus binding of arginine-rich proteins may allosterically activate PKC. Selective stimulation of PKC by arginine-rich proteins may be a mechanism by which protamine can bypass activator requirements. Furthermore, control of PKC activity by activator-independent binding of arginine-rich polypeptides suggests that altering access to certain cellular proteins may be a mechanism for PKC regulation in vivo.
蛋白激酶C（PKC）的蛋白质底物具有磷酸化结构域，这些结构域通常富含碱性氨基酸精氨酸和赖氨酸。然而，富含精氨酸的蛋白质与PKC的相互作用可能与富含赖氨酸的蛋白质不同，即富含赖氨酸的组蛋白需要磷脂和Ca2+才能被磷酸化，而富含精氨酸的蛋白质鱼精蛋白可以绕过这些效应物需求。我们研究了PKC与鱼精蛋白、组蛋白、聚-L-精氨酸和聚-L-赖氨酸的相互作用，以更好地理解碱性蛋白质结构域在PKC激活、效应物依赖性和底物特异性中的作用。使用微量滴定板结合试验，发现在没有磷脂、Ca2+和MgATP的情况下，PKC与鱼精蛋白和聚-L-精氨酸紧密结合，但不与组蛋白或聚-L-赖氨酸结合。此外，聚-L-精氨酸在抑制鱼精蛋白磷酸化方面比聚-L-赖氨酸有效得多；即1-2 nM聚-L-精氨酸足以导致50%的鱼精蛋白磷酸化抑制，而超过300 microM聚-L-赖氨酸才能达到50%的抑制。在没有激活剂的情况下，PKC的自磷酸化受到鱼精蛋白和聚-L-精氨酸的强烈刺激，但不受组蛋白或聚-L-赖氨酸的刺激，这表明富含精氨酸的多肽对PKC有选择性刺激。使用同工酶混合物（α/β/γ）或分离的PKC-β对鱼精蛋白磷酸化的双倒数图呈抛物线形，对速度/[鱼精蛋白]与速度图的动力学数据分析表明，鱼精蛋白具有正协同性。这些发现与自磷酸化实验的结果一致，即PKC似乎受到富含精氨酸的多肽的选择性刺激。这些结果表明，在没有磷脂和Ca2+的情况下，PKC可以优先结合富含精氨酸的蛋白质。这种相互作用似乎位于催化位点的远端，因此富含精氨酸的蛋白质的结合可能会变构激活PKC。富含精氨酸的蛋白质对PKC的选择性刺激可能是鱼精蛋白能够绕过激活剂需求的一种机制。此外，通过富含精氨酸的多肽的非激活剂依赖性结合来控制PKC活性表明，改变对某些细胞蛋白质的接触可能是体内PKC调节的一种机制。
