In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction
In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction
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肌球蛋白磷酸酶靶向亚基 1 磷酸化位点 T694 和 T852 在膀胱平滑肌收缩中的体内作用
DOI：
10.1113/jphysiol.2014.283853
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发表时间：
2015-02-01
影响因子：
5.5
通讯作者：
Zhu, Min-Sheng
中科院分区：
文献类型：
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作者：
Chen, Cai-Ping;Chen, Xin;Zhu, Min-Sheng
平滑肌中力的产生和维持在很大程度上由肌球蛋白调节轻链（RLC）磷酸化控制，其依赖于Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶（MLCP）活性之间的平衡。MYPT 1是MLCP的调节亚基，其在体外通过T694或T852磷酸化生化抑制MLCP活性。在此，我们分别通过建立两个单点突变小鼠系T694 A和T852 A来研究这两个磷酸化位点在膀胱平滑肌中的贡献，并且发现MYPT 1 T694的磷酸化而不是T852的磷酸化通过抑制MLCP活性和增强RLC磷酸化来介导体内的力维持。我们的研究结果揭示了MYPT 1 T694/T852磷酸化在体内调节平滑肌收缩中的作用，平滑肌中力的产生和维持在很大程度上由不同的信号模块控制，这些信号模块微调肌球蛋白调节轻链（RLC）磷酸化，而RLC磷酸化依赖于Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶（MLCP）活性之间的平衡。为了研究MLCP活性在体内的调节，我们分析了MYPT 1（MLCP的调节亚基）上的两个磷酸化位点在体外生化抑制MLCP活性的作用。MYPT 1在T694位点被体内未鉴定的激酶组成性磷酸化，而T852位点被RhoA相关蛋白激酶（ROCK）磷酸化。我们建立了T694或T852丙氨酸取代的两个小鼠系。从T852 A小鼠分离的膀胱平滑肌显示RLC磷酸化或力反应没有显着变化，但力与ROCK抑制剂抑制。相反，含有T694 A突变的平滑肌显示力的显著降低沿着RLC磷酸化的降低。T694 A突变体平滑肌的收缩反应也不依赖于ROCK激活。因此，MYPT 1 T694而不是T852的磷酸化是有助于体内MLCP活性抑制和RLC磷酸化增强的主要机制。MYPT 1 T694的组成性磷酸化可能提供调节平滑肌的力维持的机制。
Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.