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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1657.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

PYK2/PDZ-RhoGEF Links Ca²⁺ Signaling to RhoA

Zhekang Ying; Fernanda R.C. Giachini; Rita C. Tostes; R. Clinton Webb

From the Department of Physiology (Z.Y., F.R.C.G., R.C.T., R.C.W.), Medical College of Georgia, Augusta; and Pharmacology, Institute of Biomedical Sciences (F.R.C.G., R.C.T.), University of Sao Paulo, Brazil.

Correspondence to Zhekang Ying, Davis Heart & Lung Research Institute, Ohio State University, BLDG BRT/RM350, 460 W 12th Avenue, Columbus, OH 43210. E-mail zhekang.ying{at}osumc.edu

Objective— Ras homolog gene family member A (RhoA)/Rho-kinase-mediated Ca²⁺ sensitization is a critical component of constrictor responses. The present study investigates how angiotensin II activates RhoA.

Methods and 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 Ca²⁺ ionophore, demonstrating that PYK2/PDZ-RhoGEF couples RhoA activation to Ca²⁺.

Conclusions— PYK2 and PDZ-RhoGEF are necessary for angiotensin II–induced RhoA activation and for Ca²⁺ signaling to RhoA.

The mechanism by which vasoconstrictors activate RhoA remains elusive. In the present study, we show that in response to angiotensin II, PYK2 is activated and subsequently phosphorylates PDZ-RhoGEF. The PYK2/PDZ-RhoGEF pathway is sufficient to couple Ca²⁺ signaling to RhoA, thus offering a mechanism involved in constrictor responses.

Key Words: angiotensin II • RhoA • Ca²⁺ sensitization • PDZ-RhoGEF • PYK2