ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.

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Dexamethasone, a synthetic glucocorticoid, is often used to induce osteoblast commitment of mesenchymal stem cells (MSCs), and this process requires RhoA-dependent cellular tension. The underlying mechanism is unclear. In this study, we show that dexamethasone stimulates expression of fibronectin and integrin α5 (ITGA5), accompanied by an increase in the interaction of GEF-H1 (also known as ARHGEF2) with Sec5 (also known as EXOC2), a microtubule (MT)-regulated RhoA activator and a component of the exocyst, respectively. Disruption of this interaction abolishes dexamethasone-induced cellular tension and GEF-H1 targeting to focal adhesion sites at the cell periphery without affecting dexamethasone-induced levels of ITGA5 and fibronectin, and the extracellular deposition of fibronectin at adhesion sites is specifically inhibited. We demonstrate that dexamethasone stimulates the expression of serum-glucocorticoid-induced protein kinase 1 (SGK1), which is necessary and sufficient for the induction of the Sec5-GEF-H1 interaction. Given the function of SGK1 in suppressing MT growth, our data suggest that the induction of SGK1 through treatment with dexamethasone alters MT dynamics to increase Sec5-GEF-H1 interactions, which promote GEF-H1 targeting to adhesion sites. This mechanism is essential for the formation of fibronectin fibrils and their attachment to integrins at adhesion sites in order to generate cellular tension.

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