New Frontier of molecular mechanism of vascular smooth muscle contraction.

肌球蛋白轻链激酶(SmMLCK)是钙调蛋白依赖的蛋白激酶家族，催化肌球蛋白调节轻链(MLC20)的磷酸化，在激活肌球蛋白连接的收缩中起重要作用。此外，SmMLCK还具有肌动蛋白结合活性和肌球蛋白结合活性。这些被称为非激活酶活性。前者抑制肌球蛋白ATPase活性，后者激活。为了阐明这些分子机制，我们尝试在大肠杆菌中制备重组的SmMLCK。利用冷休克启动子的新型细菌表达系统，成功地实现了全长重组SmMLCK的高效表达(5-10 mg/L)。重组SmMLCK是一种可溶性形式，它能磷酸化MLc20。它还具有非激酶活性，如肌动蛋白结合和捆绑活性。具有SmMLCK特性的重组SmMLCK得到了定量表达，为进一步研究MLCK的结构和功能奠定了基础。也有报道称，SmMLCK受多种激酶的调节，但目前尚不清楚上游信号如何调节SmMLCK。我们发现，从牛胃中纯化的SmMLCK已经被内源性激酶部分磷酸化。通常情况下，重组的SmMLCK在细菌中不被磷酸化。因此，我们利用重组的SmMLCK和不同的Ser/Thr激酶来检测哪种类型的激酶使MLCK磷酸化。重组SmMLCK被MAPK、PAK、PKA、PKC和ROCK1在无钙/钙调蛋白的情况下磷酸化。在钙/钙调蛋白存在下，除PKA外，其他激酶均不能整合到重组SmMLCK中。我们用4000Q Trap LC/MS/MS测定了这些磷酸化位点，这些上游酶对MLCK的磷酸化可能在调节肌动球蛋白连接的平滑肌细胞收缩中具有重要的生理学意义。

Smooth muscle myosin light chain kinase (SmMLCK), family of calmodulin-dependent protein kinases, catalyzes the phosphorylation of regulatory light chain (MLc 20) of myosin, and plays an important role in activating actomyosin-linked contractility in smooth muscle cell. SmMLCK also exhibits actin-binding and myosin-binding activities in addition to this kinase activity. These are called non-kinase activity. The former inhibits myosin ATPase activity, and latter activates. In order to clarify these molecule mechanisms, we tried making the recombinant SmMLCK in E.coli. We succeeded in the high-level expression (5-10 mg/L culture medium) of full-length recombinant SmMLCK by new bacterial expression system using the cold shock promoter. Recombinant SmMLCK was a soluble form and it phosphorylated MLc20. It also had non-kinase activity such as actin-binding and -bundling activities. Recombinant SmMLCK, with the properties known for SmMLCK, was quantifiably expressed and is the first step in analyzing the structure and function of MLCK. It is also reported that SmMLCK is regulated by several kinases, but it is not clear how upstream signaling regulates SmMLCK. We found that the purified SmMLCK from the bovine stomach was already partially phosphorylated by endogenous kinase. Usually the recombinant SmMLCK is not phosphorylated in bacteria. Therefore we examined which kind of kinase phosphorylated MLCK by using the recombinant SmMLCK and various Ser/Thr kinases. Recombinant SmMLCK was phosphorylated by MAPK, PAK, PKA, PKC and ROCK1 phosphorylated in the absence of Ca/calmodulin. Except for the PKA, other kinase could not incorporate into recombinant SmMLCK in the presence of Ca/calmodulin. We determined these phosphorylation sites by the 4000 Q Trap LC/MS/MS. The phosphorylation of MLCK by those upstream kinases may be physiologically important in regulating of actomyosin-linked contractility in smooth muscle cell.