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 Iα (PKGIα). 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 PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα 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 PKGIα (residues 1-59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.
Molecular Cardiology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Mikio
M
Kato
Robert
R
Blanton
Guang-Rong
GR
Wang
Timothy J
TJ
Judson
Yuich
Y
Abe
Masafumi
M
Myoishi
Richard H
RH
Karas
Michael E
ME
Mendelsohn
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D001011:Aorta / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D006973:Hypertension / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C066737:RHOA protein, human; C515851:ROCK1 protein, human; C515854:Rock1 protein, mouse; D054460:rho-Associated Kinases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein