Direct association of RhoA with specific domains of PKC-

Pang, Haiyan, and Khalil N. Bitar. Direct association of RhoA with specific domains of PKC. Am J Physiol Cell Physiol 289: C982–C993, 2005. First published June 1, 2005; doi:10.1152/ajpcell.00364.2004.— Previous studies performed at our laboratory have shown that agonistinduced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKCinvolved in direct interaction with RhoA, His-tagged PKCproteins of individual domains and different combinations of PKCdomains 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 PKCin this study. The data indicate that RhoA directly bound to full-length PKC, both in vitro (82.57 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC[PKC(C1)] (70.48 20.78% above control), PKC(C2) (72.26 29.96% above control), and PKC(C4) (90.58 26.79% above control), but not to PKC(C3) (0.64 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC, PKC(C2, C3, and C4), and PKC(C3 and C4) (94.09 12.13% and 85.10 16.16% above control, respectively), but not to PKC(C1, C2, and C3) or to PKC(C2 and C3) (0.47 1.26% and 7.45 10.76% above control, respectively). RhoA bound to PKC(C1 and C2) (60.78 13.78% above control) only in vitro, but not in transfected cells, and PKC(C2, C3, and C4) and PKC(C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC. The studies using cells transfected with truncated forms of PKCindicate that PKC(C1 and C2), PKC(C1, C2, and C3), and PKC(C2 and C3) did not associate with RhoA. Only full-length PKC, PKC(C2, C3, and C4), and PKC(C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKCwith RhoA may require the C4 domain.