New Frontier of molecular mechanism of vascular smooth muscle contraction.

平滑肌肌球蛋白轻链激酶（SmMLCK）是钙调素依赖性蛋白激酶家族，催化肌球蛋白调节轻链（MLc 20）的磷酸化，在激活平滑肌细胞肌动球蛋白相关收缩性中发挥重要作用。SmMLCK还表现出肌动蛋白结合和肌球蛋白结合活性，除了这种激酶活性。这些被称为非激酶活性。前者抑制肌球蛋白ATP酶活性，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SmMLCK。利用冷激启动子构建的新型细菌表达系统，成功地在5-10 mg/L培养基中高效表达了全长重组SmMLCK。重组SmMLCK是可溶性形式，其磷酸化MLc 20。它也有非激酶活性，如肌动蛋白结合和捆绑活动。重组SmMLCK，与已知的SmMLCK的特性，定量表达，是分析MLCK的结构和功能的第一步。也有报道称，SmMLCK受几种激酶的调节，但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，重组SmMLCK在细菌中不被磷酸化。因此，我们通过使用重组SmMLCK和各种Ser/Thr激酶来检测哪种激酶磷酸化MLCK。重组SmMLCK被MAPK、PAK、PKA、PKC磷酸化，ROCK 1在无Ca/钙调素的情况下被磷酸化。除PKA外，其它激酶在Ca/钙调素存在下不能掺入SmMLCK。我们通过4000 Q Trap LC/MS/MS确定了这些磷酸化位点。这些上游激酶对MLCK的磷酸化可能在平滑肌细胞肌动球蛋白相关收缩性的调节中具有重要的生理意义。

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.