RhoA and RhoC GTPases are 92% identical but demonstrate unique regulation and function. Phosphorylation of Ser188 has widely been reported to inhibit RhoA activity. RhoC possesses Arg188 in place of Ser188 but retains a canonical upstream PKA recognition sequence. We report here that RhoC-R188S was a PKA substrate in vitro and exhibited less GTP loading compared to wild-type RhoC when expressed in cells. Transiently expressed RhoC was found to be significantly more membrane associated than RhoA. Membrane association of RhoC-R188S and RhoC-R188A were similar to each other and wild-type RhoA, suggesting that Arg188 directly promotes RhoC membrane binding. The positive influence of Arg188 on RhoC membrane association was evident in a constitutively active (Q63L) background. In accordance, RhoA-S188R was significantly more membrane associated than either RhoA or RhoA-S188A. Altogether, these data suggest that swapping residue 188 identity effectively flips the membrane binding profile of wild-type RhoA and RhoC through positive arginine contribution rather than negative phosphoserine regulation.

Presence of a negative charge at RhoA residue 188, such as through PKA‐catalyzed serine phosphorylation, has been found to destabilize RhoA membrane association. Closely related RhoC retains an adjacent canonical PKA recognition sequence (KRR), but possesses an Arg residue in place of serine at position 188. Therefore, we propose divergence at residue 188 between these two Rho isoforms directly impacts membrane association. We found transiently expressed RhoC more membrane associated than RhoA when expressed in ovarian cancer cells. Using a residue swap approach, RhoA‐S188R was found more membrane associated than wild‐type RhoA, while RhoC‐R188S had similar membrane association as wild‐type RhoC. At the same time, RhoC‐R188S was more effectively driven from membranes following forskolin treatment than RhoC. Altogether, these data suggest that a positively charged residue at position 188 strengthens RhoC membrane association and provides a mechanism for regulatory divergence.