MUTAGENESIS OF THE PHOSPHORYLATION SITE (SERINE-19) OF SMOOTH-MUSCLE MYOSIN REGULATORY LIGHT-CHAIN AND ITS EFFECTS ON THE PROPERTIES OF MYOSIN
MUTAGENESIS OF THE PHOSPHORYLATION SITE (SERINE-19) OF SMOOTH-MUSCLE MYOSIN REGULATORY LIGHT-CHAIN AND ITS EFFECTS ON THE PROPERTIES OF MYOSIN
复制标题
DOI：
10.1021/bi00169a027
复制
发表时间：
1994-01-25
期刊：
影响因子：
2.9
通讯作者：
IKEBE, M
中科院分区：
文献类型：
--
作者：
KAMISOYAMA, H;ARAKI, Y;IKEBE, M
获得了平滑肌调节轻链的全长cDNA，并在大肠杆菌表达系统中表达了重组调节轻链。使用亚基交换策略将重组调节轻链引入肌球蛋白或HMM中[Morita，J.，Takashi，R.，& Ikebe，M.（1991）Biochemistry 30，9539-9545]。重组野生型调节轻链表现出与天然分离物相同的生物学特性，即，肌球蛋白轻链激酶在Ser-19的磷酸化和磷酸化激活的肌动球蛋白ATP酶活性。为了阐明ATP酶通过磷酸化的活化是否仅仅是由于引入负电荷，我们产生了三种突变轻链。其中两个含有Ser-19被Asp或Ala取代，第三个含有Asp取代Thr-18和Ser-19。掺入的Asp突变体部分激活肌动球蛋白ATP酶的活性，但激活水平显着低于磷酸化。Asp/Asp突变体进一步激活肌动球蛋白ATP酶活性。另一方面，Ala突变体不影响ATP酶活性。Asp突变体的掺入对肌球蛋白10 S-6S构象转变和肌丝形成影响不大。Asp/Asp突变体对肌球蛋白10 S-6S构象转变和肌丝形成的影响更为显著。这些结果表明，平滑肌肌球蛋白的激活需要在特定的空间位置引入负电荷。使用Ser-19缺陷突变体，还研究了Thr-18磷酸化对肌球蛋白功能的影响。肌动蛋白激活的肌球蛋白ATP酶活性显着激活的Thr-18的磷酸化。Thr-18磷酸化也稳定了肌球蛋白的6S构象，并诱导肌球蛋白丝的形成。
A full-length cDNA Of smooth Muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system. The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy [Morita, J., Takashi, R., & Ikebe, M. (1991) Biochemistry 30, 9539-9545]. The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity. To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains. Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19. Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation. The Asp/Asp mutant further activated actomyosin ATPase activity. On the other hand, the Ala mutant did not affect the ATPase activity. Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin. The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin. These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position. Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied. Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18. The Thr-18 phosphorylation also stabilized the 6S conformation of myosin and induced myosin filament formation.