Direct association of RhoA with specific domains of PKC-α

Abstract

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