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.
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.
Yazaki
Akira
A
The 2nd Department of Internal Medicine, Mie University School of Medicine, Mie 514-8507, Japan.
Tamaru
Satoshi
S
Sasaki
Yasuharu
Y
Komatsu
Nobuo
N
Wada
Hideo
H
Shiku
Hiroshi
H
Nishikawa
Masakatsu
M
D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007938:Leukemia; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000096:biosynthesis; D043263:Myosin-Light-Chain Phosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D021122:Protein Subunits; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D013917:Thrombin