Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C
Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C
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DOI：
10.1074/jbc.272.29.18382
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Newton, AC
中科院分区：
文献类型：
--
作者：
Edwards, AS;Newton, AC
成熟的蛋白激酶C在保守的羧基末端基序处磷酸化，该基序包含由两个疏水性残基包围的Ser（或Thr）;在蛋白激酶C β II中，该残基是Ser-660（Keranen，L. M.，Dutil，E. M.，和Newton，A. C.（1995）Curr. 5，1394-1403），该贡献考察了在该位置的负电荷如何调节蛋白激酶C的功能。具体地说，蛋白激酶C β II中的Ser-660突变为Ala或Glu，并且将该酶的稳定性、膜相互作用、Ca 2+调节和动力学参数与在残基660处磷酸化的野生型蛋白质进行比较，在该位置处的负电荷对该酶的二酰基甘油刺激的膜相互作用以及伴随膜结合的构象变化没有显著影响。相比之下，磷酸盐导致酶对Ca 2+的亲和力增加10倍，对磷脂酰丝氨酸的亲和力增加相当大，这两种相互作用是由C2结构域介导的。负电荷还增加了蛋白质的热稳定性，并降低了其对ATP和肽底物的Km。这些数据表明，在蛋白激酶C的羧基末端的磷酸化结构的活性位点，使其结合ATP和底物具有更高的亲和力和结构的决定簇的调节区，使更高的亲和力结合的Ca 2+。蛋白激酶C β II中Ser-660周围的基序在许多其他激酶中发现，表明羧基末端磷酸化促进的相互作用可能提供了稳定激酶结构的一般机制。
Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C beta II, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403), This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C beta II was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660, Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain, Negative charge also increased the protein's thermal stability and decreased its K-m, for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C beta II is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.