Inactivation of Ca2+/calmodulin-dependent protein kinase II by basal autophosphorylation.
Inactivation of Ca2+/calmodulin-dependent protein kinase II by basal autophosphorylation.
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DOI：
10.1016/s0021-9258(18)53160-4
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发表时间：
1993-04
期刊：
影响因子：
--
通讯作者：
R. Colbran
中科院分区：
文献类型：
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作者：
R. Colbran
孵育纯化的大鼠前脑Ca 2 +/钙调素依赖性蛋白激酶II（CaMKII）或纯化的重组、杆状病毒表达的CaMKII小鼠α亚基（Brickey，D.一、科尔布兰河J.，Fong，Y. L.，和Soderling，T. R.（1990）Biochem.Biophys. Res. Commun. 173，578 - 584）与EGTA、乙酸镁和[γ-32 P] ATP的反应导致在30 ℃下在60分钟内掺入高达1.0 mol的32 PO 4/mol亚基;丝氨酸和苏氨酸残基都被自磷酸化。这种基础自磷酸化的Vmax为0.051 +/-0.005 mol 32PO4/mol亚基/min，Km（ATP）为145 +/-8 microM。测定Ca2 +/钙调素依赖性自磷酸化的Vmax和Km（ATP）值分别为1.3 +/-0.4 mol/mol/min和19 +/-2 microM。基础自磷酸化导致Ca2 +/钙调素依赖性激酶活性对外源性肽底物失活; Ca（2+）-非依赖性CaMKII活性没有可测量的增加。用不可水解的ATP类似物代替ATP预孵育后未观察到灭活。CaMKII，已被灭活的基础自磷酸化可以完全重新激活与纯化的蛋白磷酸酶2A的催化亚基温育。基础自磷酸化后，钙调素结合能力的CaMKII也减少，大概占所观察到的失活。失活和减少钙调素结合，导致从基础的自磷酸化被废除的突变苏氨酸306丙氨酸，但不是由突变苏氨酸305丙氨酸。此外，苏氨酸306而不是苏氨酸305突变为丙氨酸降低了苏氨酸残基处的基础自磷酸化的程度。因此，苏氨酸306处的基础自磷酸化阻止钙调蛋白结合，导致CaMKII失活。
Incubation of either purified rat forebrain Ca2+/calmodulin-dependent protein kinase II (CaMKII) or the purified recombinant, baculovirus-expressed mouse alpha subunit of CaMKII (Brickey, D. A., Colbran, R. J., Fong, Y.-L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173, 578-584) with EGTA, magnesium acetate, and [gamma-32P]ATP resulted in the incorporation of up to 1.0 mol of 32PO4/mol of subunit within 60 min at 30 degrees C; both serine and threonine residues became autophosphorylated. The Vmax for this basal autophosphorylation was 0.051 +/- 0.005 mol of 32PO4/mol of subunit/min, and the Km(ATP) was 145 +/- 8 microM. Vmax and Km(ATP) values for Ca2+/calmodulin-dependent autophosphorylation were determined to be 1.3 +/- 0.4 mol/mol/min and 19 +/- 2 microM, respectively. Basal autophosphorylation resulted in inactivation of Ca2+/calmodulin-dependent kinase activity toward exogenous peptide substrate; there was no measurable increase in Ca(2+)-independent CaMKII activity. Inactivation was not observed following preincubation with non-hydrolyzable ATP analogs in place of ATP. CaMKII that had been inactivated by basal autophosphorylation could be fully re-activated by incubation with the purified catalytic subunit of protein phosphatase 2A. Following basal autophosphorylation, the calmodulin-binding ability of CaMKII was also reduced, presumably accounting for the observed inactivation. Both the inactivation and the decrease in calmodulin-binding that resulted from basal autophosphorylation were abrogated by mutation of threonine 306 to alanine, but not by mutation of threonine 305 to alanine. Furthermore, mutation of threonine 306, but not threonine 305, to alanine reduced the extent of basal autophosphorylation at threonine residues. Thus, basal autophosphorylation at threonine 306 blocks calmodulin binding, resulting in inactivation of CaMKII.