PYK2/PDZ-RhoGEF links Ca2+ signaling to RhoA

Abstract

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.

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 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.

Figure 1

PDZ-RhoGEF mediates RhoA activation by angiotensin II. A-C, rat VSMCs were infected with control, LARG, PDZ-RhoGEF, or p115RhoGEF adenoviral vector. After 40 hours, these cells were stimulated with angiotensin II (100 nM) for 3 minutes, and then RhoA translocation or MYPT1 phosphorylation was analyzed by western blot. A representative result (A) and the summary (B and C) of at least four independent experiments are presented. *p<0.05 vs. Vehicle infected with the same adenoviral vector; ^#p<0.05 vs. AngII-Vector; ^$p<0.05 vs. Vehicle-Vector; student's t test with bonferroni correction. D and E, rat VSMCs were infected with LARG or PDZ-RhoGEF adenoviral vector. After 40 hours, these cells were stimulated with angiotensin II (100 nM) for 3 minutes, then the subcellular localization of over-expressed LARG or PDZ-RhoGEF is analyzed by western blot with anti-HA. n = 3. *p<0.05 vs. vehicle; student's t test with bonferroni correction.

Figure 2

Knockdown of PDZ-RhoGEF reduces RhoA activation by angiotensin II. A-C, rat VSMC were infected with control or PDZ-RhoGEF siRNA adenoviral vector. After 40 hours, these cells were stimulated with angiotensin II (100 nM) for 3 minutes, then RhoA translocation or MYPT1 phosphorylation was analyzed by western blot. A representative result (A) and the summary (B-D) of at least four independent experiments are presented. ^&p<0.05 vs. control siRNA; *p<0.05 vs. vehicle infected with the same adenoviral vector; ^#p<0.05 vs. AngII-Control siRNA; student's t test with bonferroni correction. E-G, rVSMC were infected with the indicated adenoviral vectors. After 40 hours, these cells were stimulated with angiotensin II (100 nM) for 3 minutes, then RhoA translocation or MYPT1 phosphorylation was analyzed by western blot. A representative result (E) and the summary (F and G) of at least three independent experiments are presented. *p<0.05 vs. vehicle infected with the same adenoviral vector; ^#p<0.05 vs. AngII-Control siRNA; student's t test with bonferroni correction.

Figure 3

PYK2 mediates RhoA activation through phosphorlating PDZ-RhoGEF. A, rat VSMCs were stimulated with angiotensin II (100 nM) for 3 minutes. PDZ-RhoGEF was then immuno-precipitated, and analyzed by western blot. A representative result of two independent experiments was presented. B, immuno-precipitated myc-tagged PDZ-RhoGEF was incubated with constitutively active PYK2 in the phosphorylation buffer for 1 hour at 37°C, and the tyrosine phosphorylation and the GEF activity of PDZ-RhoGEF were analyzed. n≥3. * p<0.05; student's t test. C-F, rat VSMCs were infected with PYK2 siRNA adenoviral vectors. After 40 hours, cells were stimulated with angiotensin II (100 nM) for 3 minutes, and RhoA translocation or MYPT1 phosphorylation was analyzed by western blot. A representative result (C) and the summary (D-F) of 3 independent experiments are presented. ^&p<0.05 vs. control siRNA; *p<0.05 vs. vehicle infected with the same adenoviral vector; ^#p<0.05 vs. AngII-Control siRNA; student's t test with bonferroni correction. G, rat VSMCs were infected with control, PYK2, or PYK2 combined with PDZ-RhoGEF siRNA adenoviral vector. After 40 hours, cells were harvested, and RhoA translocation or MYPT1 phosphorylation was analyzed by western blot.

Figure 4

PYK2/PDZ-RhoGEF couples Ca2+ signaling to RhoA. A-C, rat VSMCs were infected with control, PYK2 or PDZ-RhoGEF siRNA adenoviral vectors. After 40 hours, cells were stimulated with ionomycine (1 μM) for 30 minutes, and RhoA translocation or MYPT1 phosphorylation was analyzed by western blot. A representative result (A) and the summary (B and C) of at least three independent experiments are presented. *p<0.05 vs. vehicle infected with the same adenoviral vector; ^#p<0.05 vs. AngII-Control siRNA; student's t test with bonferroni correction. D, The expression level of PYK2 and PDZ-RhoGEF in the indicated cell lines was analyzed by western blot. After stimulation with ionomycine (1 μM) for 30 minutes, the GTP bound RhoA was measured by G-LISA™ Small G-protein Activation Assays. The summary of three independent experiments is shown. *p<0.05; student's t test. E, COS-7 or NIH-3T3L1 cells were infected with the indicated adenoviral vectors. After 40 hours, RhoA translocation was measured by western blot. A representative result of two independent experiments is shown.