Direct association of RhoA with specific domains of PKC-α

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)-alpha and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC-alpha involved in direct interaction with RhoA, His-tagged PKC-alpha proteins of individual domains and different combinations of PKC-alpha domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-alpha in this study. The data indicate that RhoA directly bound to full-length PKC-alpha, both in vitro (82.57 +/- 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC-alpha [PKC-alpha (C1)] (70.48 +/- 20.78% above control), PKC-alpha (C2) (72.26 +/- 29.96% above control), and PKC-alpha (C4) (90.58 +/- 26.79% above control), but not to PKC-alpha (C3) (0.64 +/- 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-alpha, PKC-alpha (C2, C3, and C4), and PKC-alpha (C3 and C4) (94.09 +/- 12.13% and 85.10 +/- 16.16% above control, respectively), but not to PKC-alpha (C1, C2, and C3) or to PKC-alpha (C2 and C3) (0.47 +/- 1.26% and 7.45 +/- 10.76% above control, respectively). RhoA bound to PKC-alpha (C1 and C2) (60.78 +/- 13.78% above control) only in vitro, but not in transfected cells, and PKC-alpha (C2, C3, and C4) and PKC-alpha (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-alpha. The studies using cells transfected with truncated forms of PKC-alpha indicate that PKC-alpha (C1 and C2), PKC-alpha (C1, C2, and C3), and PKC-alpha (C2 and C3) did not associate with RhoA. Only full-length PKC-alpha, PKC-alpha (C2, C3, and C4), and PKC-alpha (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-alpha with RhoA may require the C4 domain.