Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase
Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase
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DOI：
10.1042/bj20020401
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发表时间：
2002-08-15
影响因子：
4.1
通讯作者：
Hartshorne, DJ
中科院分区：
文献类型：
--
作者：
Murányi, A;MacDonald, JA;Hartshorne, DJ
肌球蛋白磷酸酶（MP）活性的调节机制是肌球蛋白磷酸酶靶亚基（MYPT 1）的磷酸化。整合素连接激酶（ILK）与收缩机制相关，可在肌球蛋白轻链激酶位点磷酸化肌球蛋白。ILK也可能磷酸化和调节MP的可能性进行了研究。蛋白质印迹和凝胶内激酶测定显示ILK与MP全酶相关。MYPT 1被ILK磷酸化，并在MYPT 1的N-和C-末端片段中检测到磷酸化位点。从序列分析中，确定了三个位点：一个主要位点在Thr（709），另外两个位点在Thr（695）和Thr（495）。cAMP依赖性蛋白激酶（PKA）的一个位点是Ser（694）。与I型磷酸酶的催化亚基的分析表明，只有C-末端片段的MYPT 1的拉链相互作用的蛋白激酶磷酸化，ILK抑制活性。磷酸化的N-末端片段激活磷酸酶活性和PKA磷酸化没有影响。使用被各种激酶磷酸化的全长MYPT 1构建体，显示Rho激酶产生显著的抑制; ILK产生中等水平的抑制，其对于Thr（695）-> Ala突变体显著降低; PKA没有影响。总之，各种位点的磷酸化表明，Thr（695）是主要的抑制位点，Thr（709）仅具有轻微的抑制作用，Ser（694）没有作用。ILK磷酸化MYPT 1和肌球蛋白以及ILK与MP的关联表明ILK可能影响细胞骨架结构或功能。
A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type I phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695) --> Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.