Characterization of the mechanism of regulation of Ca2+/calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase
Characterization of the mechanism of regulation of Ca2+/calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase
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DOI：
10.1074/jbc.273.34.21473
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发表时间：
1998-08-21
影响因子：
4.8
通讯作者：
Nairn, AC
中科院分区：
文献类型：
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作者：
Matsushita, M;Nairn, AC
Ca 2 +/钙调素依赖性蛋白激酶I（CaMKI）通过COOH末端螺旋-环-螺旋（Ile（286）-Met（316））调节结构域与催化核心的相互作用维持在自抑制状态。钙调蛋白（CaM）对酶的激活也允许CaMKI被磷酸化并被第二种酶CaMK激酶（CaMKK）激活。为了更彻底地表征CaM对CaMKI的调节及其与CaMKK磷酸化的相互关系，我们使用重组野生型（WT）和突变形式的CaMKI和CaMKK进行了详细的结构-功能分析。CaMKI-WT，在不存在CaM的情况下，或CaMKI-299和CaMKI-298被自身抑制，并且不能被CaMKK-433（CaMKK的截短的组成型活性形式）磷酸化。去除Phe（298）（CaMK-297）产生CaMKI的组成型活性形式，其也被CaMKK-433磷酸化。CaMKI-WT在不存在CaM的情况下基本上无活性（对于通过CaM的活化，K = 0.5，类似于30 nM）。Ile（294）和Phe（298）突变为丙氨酸（CaMKI-2A）导致可测量的基础酶活性。Ile（286）和瓦尔（290）突变为丙氨酸（CaMKI-4A）增加了该基础活性。Trp（303）（CaMKI-W303 S）的突变导致CaM的K-0.5的大幅增加（类似于100 μ M），支持该残基作为CaM的初始靶标的作用。Phe（307）（CaMKI-F307 A）突变导致基础酶活性增加，支持该残基在CaMKI自抑制中的作用。这些研究共同证明了特定氨基酸在CaMKI的自抑制中以及在其被CaM激活和被CaMKK磷酸化中的关键作用。
Ca2+/calmodulin-dependent protein kinase I (CaMKI) is maintained in an autoinhibited state by the interaction of a COOH-terminal helix-loop-helix (Ile(286)-Met(316)) regulatory domain with the catalytic core. Activation of the enzyme by calmodulin (CaM) also allows CaMKI to be phosphorylated and activated by a second enzyme, CaMK kinase (CaMKK). To more thoroughly characterize the regulation of CaMKI by CaM and its interrelationship with phosphorylation by CaMKK, we have carried out a detailed structure-function analysis using recombinant wild-type (WT) and mutant forms of CaMKI and CaMKK. CaMKI-WT, in the absence of CaM, or CaMKI-299 and CaMKI-298 were autoinhibited and could not be phosphorylated by CaMKK-433 (a truncated constitutively active form of CaMKK). Removal of Phe(298) (CaMK-297) generated a constitutively active form of CaMKI that was also phosphorylated by CaMKK-433. CaMKI-WT was essentially inactive in the absence of CaM (K-0.5 for activation by CaM similar to 30 nM). Mutation of Ile(294) and Phe(298) to alanine (CaMKI-2A) resulted in measurable basal enzyme activity. Additional mutation of Ile(286) and Val(290) to alanine (CaMKI-4A) increased this basal activity. Mutation of Trp(303) (CaMKI-W303S) resulted in a large increase in the K-0.5 for CaM (similar to 100 mu M), supporting a role for this residue as an initial target for CaM. Mutation of Phe(307) (CaMKI-F307A) resulted in increased basal enzyme activity, supporting a role for this residue in autoinhibition of CaMKI. Together these studies demonstrate the critical role of specific amino acids in the autoinhibition of CaMKI and also in its activation by CaM and phosphorylation by CaMKK.