Cdk5-dependent regulation of Rho activity, cytoskeletal contraction, and epithelial cell migration via suppression of Src and p190RhoGAP.
Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.
Tripathi
Brajendra K
BK
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, 5635 Fishers Lane, Room 1127, Bethesda, MD 20892, USA.
Zelenka
Peggy S
PS
D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007701:Kinetin / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
C074560:ARHGAP35 protein, human; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C090046:olomoucine; D019061:src-Family Kinases; D051360:Cyclin-Dependent Kinase 5; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins; D007701:Kinetin