Generation of Autonomous Activity of Ca2+/Calmodulin-Dependent Protein Kinase Kinase β by Autophosphorylation

被引:26
|
作者
Tokumitsu, Hiroshi [1 ]
Hatano, Naoya [2 ]
Fujimoto, Tomohito [1 ]
Yurimoto, Said [1 ]
Kobayashi, Ryoji [1 ]
机构
[1] Kagawa Univ, Fac Med, Dept Signal Transduct Sci, Kagawa 7610793, Japan
[2] Kobe Univ, Grad Sch Med, Integrated Ctr Mass Spectrometry, Kobe, Hyogo 6500017, Japan
关键词
CAM-KINASE; REGULATORY MECHANISM; MOLECULAR-CLONING; UPSTREAM KINASE; I CASCADE; CALMODULIN; ACTIVATION; CALCIUM; INHIBITION; COMPLEX;
D O I
10.1021/bi201005g
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 kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca2+ signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKK alpha and beta isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca2+/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in GaMKK beta, but phosphorylation of the equivalent Thr residue (Thr(446)) in the alpha isoforrn was not observed. Unlike CaMKK alpha that has high Ca2+/CaM-dependent activity, wild-type CaMKK beta displays enhanced autonomous activity (Ca2+/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca2+/CaM binding. In addition, a CaMKK alpha mutant containing the CaMKK beta regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the alpha isoform, CaMKK beta exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.
引用
收藏
页码:8193 / 8201
页数:9
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