Inhibition by Rho-kinase and protein kinase C of myosin phosphatase is involved in thrombin-induced shape change of megakaryocytic leukemia cell line UT-7/TPO.
Inhibition by Rho-kinase and protein kinase C of myosin phosphatase is involved in thrombin-induced shape change of megakaryocytic leukemia cell line UT-7/TPO.
复制标题
肌球蛋白磷酸酶的 Rho 激酶和蛋白激酶 C 的抑制参与了凝血酶诱导的巨核细胞白血病细胞系 UT-7/TPO 的形状变化。
DOI：
10.1016/j.cellsig.2004.07.009
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发表时间：
2005
影响因子：
4.8
通讯作者：
M. Nishikawa
中科院分区：
文献类型：
--
作者：
A. Yazaki;S. Tamaru;Y. Sasaki;N. Komatsu;H. Wada;H. Shiku;M. Nishikawa
凝血酶诱导UT-7/TPO，一个血小板生成素依赖的人巨核细胞系的形状改变。肌球蛋白轻链（MLC）激酶在UT-7/TPO细胞中的表达可忽略不计，而Rho激酶和蛋白激酶C（PKC）在UT-7/TPO细胞中可检测到。凝血酶刺激20 kDa MLC的Ser19单磷酸化和Thr18和Ser19二磷酸化，以及肌球蛋白结合亚基（MBS）和PKC增强的抑制性磷酸化蛋白肌球蛋白磷酸酶（CPI）的磷酸化。Rho激酶抑制剂Y-27632 [（+）-（R）-trans-（1-aminoethyl）-N-（4-phynidyl）cyclohexane-carboxamide dihydrochloride，monohydride]强烈抑制凝血酶诱导的形状变化、MBS磷酸化以及MLC的单磷酸化和二磷酸化。PKC抑制剂GF109203 X（2-[1-（3-二甲基氨基丙基）-1H-吲哚-3-基]-3-（1H-吲哚-3-基）-马来酰亚胺）部分抑制凝血酶诱导的形状变化和MLC二磷酸化，即使在完全抑制凝血酶诱导的CPI磷酸化的浓度下。在凝血酶诱导的UT-7/TPO细胞形态改变中，磷酸化的MBS和CPI与二磷酸化的MLC共定位于伪足，而单磷酸化的MLC主要定位于皮层区域。通过用Y-27632或GF109203X预孵育来阻断二磷酸化MLC的积累。这些结果表明，Rho激酶是负责诱导MLC磷酸化在凝血酶诱导的UT-7/TPO细胞的形状改变，肌球蛋白磷酸酶的失活，通过Rho激酶-MBS和PKC-CPI途径可能是必要的，以增强MLC的二磷酸化，促进伪足的形成。
Thrombin induced a shape change of UT-7/TPO, a thrombopoietin-dependent human megakaryocytic cell line. Expression of myosin light chain (MLC) kinase was negligible in UT-7/TPO cells, while Rho-kinase and protein kinase C (PKC) were detected. Thrombin stimulated both monophosphorylation at Ser19 and diphosphorylation at Thr18 and Ser19 of 20 kDa MLC, as well as phosphorylation of myosin-binding subunit (MBS) and PKC-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI). The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-(1-aminoethyl)-N-(4-phynidyl) cyclohexane-carboxamide dihydrochloride, monohydrade] strongly inhibited thrombin-induced shape change, MBS phosphorylation, and mono- and diphosphorylation of MLC. The PKC inhibitor GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) partially inhibited thrombin-induced shape change and MLC diphosphorylation even at the concentration that completely inhibited thrombin-induced CPI phosphorylation. In shape-changed UT-7/TPO cells induced by thrombin, phosphorylated MBS and CPI were colocalized with diphosphorylated MLC at pseudopods, whereas monophosphorylated MLC was mainly located in the cortical region. The accumulation of diphosphorylated MLC was blocked by preincubation with either Y-27632 or GF109203X. These results suggest that Rho-kinase is responsible for the induction of MLC phosphorylation in thrombin-induced shape change of UT-7/TPO cells and that myosin phosphatase inactivation through Rho-kinase-MBS and PKC-CPI pathways could be necessary for enhancement of MLC diphosphorylation which promote the pseudopod formation.