, Volume 45, Issue 3, pp 243-254

Dynamics of RhoA and ROKα translocation in single living cells

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Abstract

The RhoA-binding kinase (ROK) is one of the target kinases of RhoA and is known to play a critical role in regulating cytoskeletal rearrangement in cells. ROK translocates to the plasma membrane fraction; however, the mechanism of the translocation of ROK still remains obscure. To clarify the molecular mechanisms of the translocation of ROK, we co-transfected MDCK cells wity cyan fluorescent protein-tagged RhoA and yellow fluorescent protein-tagged ROKα, or their variants, and monitored the localization and translocation of the two different fluorescent tagged-molecules in single living cells during epithelial growth factor (EGF) stimulation. Both RhoA (wild-type) and ROKα (wild-type) translocated to ruffling membrane with EGF stimulation in several minutes. A ROKα mutant, in which Rho-binding ability is disrupted, is unable to translocate to the membrane with RhoA. However, RhoA mutant Q63L/C190R, an active form lacking membrane localization activity, abolished the translocation of wild-type ROKα, suggesting that the translocation of RhoA is critical for ROK translocation to the membrane. Another mutant lacking the pleckstrin homology domain failed in translocation as well. On the other hand, it was surprising that the kinase dead mutant succeeded in translocation to the membrane after EGF stimulation. Based on these results, we propose the following ROKα translocation mechanism. ROKα binds to RhoA in cytosol and translocates to the membrane based on the membrane-targeting ability of active RhoA. After ROKα associates with the membrane, the pleckstrin homology domain provides the stability of ROKα on the membrane. The activation of enzymatic activity or adenosine triphosphate binding, however, is not directly related to the translocation mechanism, although we found that the membrane association is critical for the activation of the kinase activity.