Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase.
Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase.
复制标题
肌球蛋白轻链激酶将第一个钙结合袋中的钙调蛋白磷酸化。
DOI：
10.1006/abbi.1996.0321
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Garcia,JG
中科院分区：
文献类型：
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作者：
Davis,HW;Crimmins,DL;Thoma,RS;Garcia,JG
在平滑肌和特定的非肌肉细胞中，肌球蛋白轻链激酶（MLCK）对调节性肌球蛋白轻链的磷酸化是肌动蛋白诱导的肌球蛋白ATP酶激活和随后的收缩事件的必要步骤。我们先前已经证明，被酪蛋白激酶II磷酸化的CaM不能激活牛血小板MLCK（Sackset al.（1992）Biochem.J.283，21-24）。虽然肌球蛋白轻链被认为是MLCK磷酸化活性的唯一已知底物，但我们现在发现MLCK磷酸化CaM。CaM的磷酸化依赖于碱性肽如聚-L-精氨酸（最佳碱性肽/CaM比= 0.08）的存在，并通过饱和[Ca 2 +]（K0.5= 16 μM）刺激。CaM磷酸化被一种特异性MLCK抑制剂KT 5926抑制，其剂量依赖性与抑制肌球蛋白轻链磷酸化的剂量依赖性相同。在激活MLCK磷酸化肌球蛋白轻链方面，天然和MLCK磷酸化CaM是不可区分的。有趣的是，MLCK中的钙调素结合位点已被删除，能够磷酸化钙调素的钙离子依赖性的方式，这表明两个钙调素分子结合到完整的MLCK同时，一个抑制（pseudosubstrate）域和一个在催化位点。MLCK的CaM磷酸化仅发生在第一个Ca 2+结合口袋中的Thr 29（90%）和Thr 26（10%）上。总之，MLCK引起的CaM磷酸化不同于其他激酶催化的CaM磷酸化（即，胰岛素受体或酪蛋白激酶II）在碱性肽和Ca 2+需求以及磷酸化位点中的作用。对该模型的进一步研究可以提供对MLCK激活和底物识别机制的深入了解。
In smooth muscle and specific nonmuscle cells the phosphorylation of the regulatory myosin light chains by myosin light chain kinase (MLCK) is an obligatory step in actin-induced activation of myosin ATPase and subsequent contractile events. We have previously demonstrated that CaM phosphorylated by casein kinase II fails to activate bovine platelet MLCK (Sackset al.(1992)Biochem. J.283, 21–24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is stimulated by saturating [Ca2+] (K0.5= 16 μM). CaM phosphorylation was inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependency identical to that for inhibition of myosin light chain phosphorylation. Native and MLCK-phosphorylated CaM were indistinguishable in activating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylate CaM in a Ca2+-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubstrate) domain and one at the catalytic site. CaM phosphorylation by MLCK occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first Ca2+-binding pocket. In summary, CaM phosphorylation by MLCK differs from CaM phosphorylation catalyzed by other kinases (i.e., the insulin receptor or casein kinase II) in both basic peptide and Ca2+requirements as well as in the sites of phosphorylation. Further investigations of this model may provide insight into the mechanisms of MLCK activation and substrate recognition.