Direct binding and regulation of RhoA protein by cyclic GMP-dependent protein kinase Iα.
J Biol Chem
; 287(49): 41342-51, 2012 Nov 30.
Article
em En
| MEDLINE
| ID: mdl-23066013
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Regulação da Expressão Gênica
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Proteínas rho de Ligação ao GTP
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Proteína rhoA de Ligação ao GTP
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Proteína Quinase Dependente de GMP Cíclico Tipo I
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos