PYK2/PDZ-RhoGEF links Ca2+ signaling to RhoA.
OBJECTIVE
Ras homolog gene family member A (RhoA)/Rho-kinase-mediated Ca(2+) sensitization is a critical component of constrictor responses. The present study investigates how angiotensin II activates RhoA.


RESULTS
Adenoviral vectors were used to manipulate the expression of regulator of G protein signaling (RGS) domain containing Rho-specific guanine exchange factors (RhoGEFs) and proline-rich tyrosine kinase 2 (PYK2), a nonreceptor tyrosine kinase, in primary rat vascular smooth muscle cells. As an evidence of RhoA activation, RhoA translocation and MYPT1 (the regulatory subunit of myosin light chain phosphatase) phosphorylation were analyzed by Western blot. Results showed that overexpression of PDZ-RhoGEF, but not p115-RhoGEF or leukemia-associated RhoGEF (LARG), enhanced RhoA activation by angiotensin II. Knockdown of PDZ-RhoGEF decreased RhoA activation by angiotensin II. PDZ-RhoGEF was phosphorylated and activated by PYK2 in vitro, and knockdown of PDZ-RhoGEF reduced RhoA activation by constitutively active PYK2, indicating that PDZ-RhoGEF links PYK2 to RhoA. Knockdown of PYK2 or PDZ-RhoGEF markedly decreased RhoA activation by A23187, a Ca(2+) ionophore, demonstrating that PYK2/PDZ-RhoGEF couples RhoA activation to Ca(2+).


CONCLUSIONS
PYK2 and PDZ-RhoGEF are necessary for angiotensin II-induced RhoA activation and for Ca(2+) signaling to RhoA.
Ying
Zhekang
Z
Department of Physiology, Medical College of Georgia, Augusta, GA, USA. zhekang.ying@osumc.edu
Giachini
Fernanda R C
FR
Tostes
Rita C
RC
Webb
R Clinton
RC
D041721:3T3-L1 Cells; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D020013:Calcium Signaling; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D051418:Focal Adhesion Kinase 2 / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D020710:RGS Proteins / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014443:Tyrosine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D020662:Guanine Nucleotide Exchange Factors; C523071:PDZ-RhoGEF, rat; D020710:RGS Proteins; D000804:Angiotensin II; D014443:Tyrosine; D051418:Focal Adhesion Kinase 2; D020742:rhoA GTP-Binding Protein