Characterization of the mechanism of regulation of Ca2+/calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase

被引:76
作者
Matsushita, M [1 ]
Nairn, AC [1 ]
机构
[1] Rockefeller Univ, Mol & Cellular Neurosci Lab, New York, NY 10021 USA
关键词
D O I
10.1074/jbc.273.34.21473
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:21473 / 21481
页数:9
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