Disruption of Rho signal transduction upon cell detachment.
Serum-soluble factors play a dominant role in the activation of the small GTPase RhoA. Cell adhesion also modulates RhoA activity but the effect is modest in the absence of serum. Here, we show that cell adhesion is required for serum-stimulated Rho signal transduction leading to myosin light chain (MLC) phosphorylation. Characterization of Rho-kinase substrates revealed that diphosphorylation of MLC at Thr-18 and Ser-19 (ppMLC(T18/S19)) and phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase at Thr-853 (pMBS(T853)) were mostly Rho and Rho-kinase dependent in attached fibroblasts. MLC monophosphorylation at Ser-19 (pMLC(S19)) was partially dependent on Rho kinase, whereas phosphorylation of MBS at Thr-696 (pMBS(T696)) and phosphorylation of CPI-17 at Thr-38 (pCPI-17(T38)) were mostly Rho-kinase independent. Cell detachment caused a significant reduction in pMLC(S19) and a more dramatic decrease of ppMLC(T18/S19) without inhibiting RhoA. pMBS(T853), pMBS(T696) and pCPI-17(T38) were not significantly reduced, suggesting that myosin-phosphatase activity was little changed. Cells expressing active RhoA (RhoA(V14)) or Rho-kinase catalytic domain maintained elevated pMBS(T853) upon detachment but failed to support ppMLC(T18/S19), indicating that the ability of Rho kinase to phosphorylate MLC is impaired. Reattachment to immobilized fibronectin resulted in a gradual recovery of Rho-kinase-induced ppMLC(T18/S19) that is absent from the cells attached to poly-L-lysine. The convergence of signals from soluble factors and cell adhesion might therefore occur at the point of MLC phosphorylation, providing an effective mechanism for dynamic control of contractility during cell migration.
Ren
Xiang-Dong
XD
Department of Dermatology, SUNY at Stony Brook, Stony Brook, NY 11794, USA. xiang-dong.ren@sunysb.edu
Wang
Ruixue
R
Li
Qinyuan
Q
Kahek
Lobna A F
LA
Kaibuchi
Kozo
K
Clark
Richard A F
RA
D001483:Base Sequence; D015153:Blotting, Western; D002448:Cell Adhesion; D017931:DNA Primers; D008856:Microscopy, Fluorescence; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D017931:DNA Primers; D018994:Myosin Light Chains; D020742:rhoA GTP-Binding Protein