Direct Binding and Regulation of RhoA Protein by Cyclic GMP-dependent Protein Kinase Iα

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase I alpha (PKGI alpha). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGI alpha and show further that PKGI alpha binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGI alpha was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1-44), containing the switch I domain of RhoA, and the amino terminus of PKGI alpha (residues 1-59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGI alpha binds RhoA, and a leucine zipper mutant PKGI alpha was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGI alpha bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGI alpha target and that direct binding of activated PKGI alpha to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.