Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading.
Cell migration and cytokinesis require reorganization of the cytoskeleton, involving phosphorylation and dephosphorylation of proteins such as myosin II and moesin. Myosin and moesin bind directly to a regulatory subunit of myosin/moesin phosphatase (MMP) that contains a protein type-1 phosphatase (PP1) catalytic subunit. Here we examined the role of MMP in cytoskeletal dynamics using a phosphorylation-dependent inhibitor protein specific for MMP, called CPI-17. Fibroblasts do not express CPI-17, making them a null background to study effects of expression. Wild type CPI-17 in rat embryo fibroblasts caused (1) abnormal accumulation of cortical F-actin fibers, distinct from the stress fibers induced by expression of active RhoA; (2) progressive contraction of cell area, leaving behind filamentous extensions that stained for F-actin and moesin, but not myosin; and (3) significantly retarded spreading of fibroblasts on fibronectin with elevated myosin II light chain phosphorylation. A phosphorylation site mutant CPI-17(T38A) and inhibitor-2 (Inh2), another PP1-specific inhibitor protein, served as controls and did not elicit these same responses when expressed at the same level as CPI-17. Inhibition of myosin light chain kinase by ML-9 prevented the abnormal accumulation of cortical microfilaments by CPI-17, but did not reverse shrinkage in area, whereas kinase inhibitors HA1077 and H7 prevented CPI-17-induced changes in microfilament distribution and cell contraction. These results highlight the physiological importance of myosin/moesin phosphatase regulation to dynamic remodeling of the cytoskeleton.
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville 22908, USA.
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Wong
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Yazawa
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Brautigan
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism; D000818:Animals; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005353:Fibronectins / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010590:Phalloidine / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D051381:Rats
D000199:Actins; D004791:Enzyme Inhibitors; D005353:Fibronectins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D010590:Phalloidine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; C049347:fasudil