Direct binding and regulation of RhoA protein by cyclic GMP-dependent protein kinase Iα

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.

FIGURE 1.

PKGI blocks LPA-induced membrane translocation of RhoA. A, representative Western blot and summary densitometry data of RhoA distribution in the membrane fraction prepared from human Ao184 vascular smooth muscle cells. Cells were treated for 30 min with control or LPA (50 μm) in the absence or presence of 8-Br-cGMP (100 μm). Cell lysates were fractionized by ultracentrifugation followed by Western blotting with RhoA antibody. B and C, distribution of RhoA in the cytosol and the membrane fractions prepared from Swiss 3T3 fibroblast cells 48 h after transient transfection with PKGIα (Β) or LZM-PKGIα (C). Summary densitometry data are shown at the right of each panel. Data are representative of four independent experiments. Error bars, S.E. *, p < 0.05 versus LPA; **, p < 0.05 versus vehicle. VSMC, A0184 vascular smooth muscle cells; ADU, arbitrary densitometric units.

FIGURE 2.

GST-RhoA interacts directly with PKGIα. Immunoblots (IB) from cell lysates (A and C) or purified PKGIα (B) are shown. GST-RhoA fusion proteins were immobilized with GSH beads and incubated for 2 h at 4 ºC with lysates from COS-1 cells transiently transfected with PKGIα (A), PKGIα purified from bovine lung (B), or lysates of COS-1 cells transiently transfected with PKGIβ (C). Co-interaction of PKGIα or Iβ with GST fusion proteins was determined by immunoblotting with anti-PKGIcom antibody. Densitometry data are shown at the right of each panel. Summary data are representative of three or four independent experiments. Error bars, S.E. *, p < 0.05; **, p < 0.01 versus control (GST alone). PKGIcom, antibody directed to common domain of PKGIα and PKGIβ. ADU, arbitrary densitometric units.

FIGURE 3.

Co-immunoprecipitation of RhoA with PKGIα in cell lysates. Immunoprecipitation (IP) of RhoA from COS-1 cells transiently transfected for 48 h with FLAG-RhoA and/or PKGIα is shown. Immunocomplexes from overnight incubation with antibody were isolated by rocking for 2 h with protein G-conjugated agarose at 4 º. Co-precipitation of PKGIα and FLAG-RhoA was determined by immunoblotting (IB) with anti-PKGIcom antibody or anti-RhoA antibody, respectively. Ig, nonspecific immunoglobulin negative control; Ab, anti-FLAG antibody. Summary densitometry data are shown at the right of the panel. Data are representative of three independent experiments. *, p < 0.05 versus control (Ig alone).

FIGURE 4.

Examination of dependence of RhoA-PKGIα interaction on RhoA nucleotide binding state. A, GST-RhoA fusion proteins were incubated with either GTPγS (denoted GTP) or GDPβS (denoted GDP), followed by incubation with purified PKGIα. Western blotting (IB) was performed with PKGcom antibody directed against the common domain of PKGIα and PKGIβ. Data are representative of three independent experiments. Error bars, S.E. *, p < 0.01 versus GST control. NS, not significant. B, GST-RhoA fusion proteins were incubated for 5 h at 4 ºC with lysates of Ao184 VSMCs, and Western blotting was performed for the RhoGDI. GST protein loading was confirmed by Ponceau S staining of the transfer membrane. C, agarose-conjugated cGMP beads were incubated for 5 h at 4 ºC with Ao184 lysates followed by Western blotting for PKGI with the PKGIcom antibody, or anti-RhoGDI. Con, agarose beads; cGMP, agarose-conjugated cGMP beads.

FIGURE 5.

Identification of the PKGIα binding domains of RhoA. A, schematic of RhoA deletion mutants used in the experiments is shown. ED, effector domain; IR, insertion region (not in Ras); HV, hypervariable region (differs between Rho/Ras isoforms). B, GST-truncated RhoA and GST-full-length RhoA fusion proteins were immobilized with GSH beads and incubated overnight at 4 ºC with PKGIα purified from bovine lung. Co-interaction of PKGIα by GST fusion proteins was determined by immunoblotting (IB) with anti-PKGIcom antibody. Summary densitometry data are shown at the bottom of the panel. Data are representative of three independent experiments. Error bars, S.E. **, p < 0.01 versus control (GST alone).

FIGURE 6.

Identification of the RhoA binding domains of PKGIα. A, schematic of PKGIα deletion mutants used in the experiments is shown. B, GST-truncated PKGIα fusion proteins were immobilized with GSH beads and incubated for 2 h at 4 ºC with 4 μg/μl His-RhoA purified from overexpressed E. coli. Co-interaction of His-RhoA by GST fusion proteins was determined by immunoblotting (IB) with anti-RhoA antibody.

FIGURE 7.

PKGIα activation by cGMP mediates RhoA-PKGIα binding. Co-expression of PKGIα and FLAG-RhoA in COS-1 cells lysed 48 h after transient transfection (A and B) or mixture of purified PKGIα and His-RhoA (C and D), followed by isolation on cGMP agarose or Rp-8AET-GMPS agarose for 2 h at 4 ºC is shown. Co-incubation of proteins was for 2 h at 4 ºC (A and B) or overnight at 4 ºC (C and D). RhoA and PKGIα complexes were detected by immunoblotting (IB) with anti-RhoA or anti PKGIcom antibody, respectively. Summary densitometry data are shown at the right of each panel. Data are representative of four independent experiments. Error bars, S.E. **, p < 0.01 versus control (agarose alone). Agarose, agarose beads alone; cGMP, cGMP-conjugated agarose beads; RP, Rp-8AET-cGMPS-conjugated agarose beads. ADU, arbitrary densitometric units.

FIGURE 8.

Requirement of functional PKGIα leucine zipper domain for RhoA interaction with activated PKGIα. A, co-expression of FLAG-RhoA and PKGIα or LZM-PKGIα in COS-1cells, followed by isolation on cGMP agarose for 2 h at 4 ºC. RhoA and PKGIα complexes were detected by immunoblotting (IB) with anti-RhoA, anti-PKGIcom, anti-PKGIα, or anti-LZM-PKGIα antibody, respectively. B, summary densitometry data. Data are representative of three independent experiments. Error bars, S.E. *, p < 0.01 versus PKGIα. Agarose, agarose beads alone; cGMP, cGMP-conjugated agarose beads. ADU, arbitrary densitometric units.

FIGURE 9.

PKGIα catalytic activity is required to inhibit RhoA membrane anchoring, but not for RhoA-PKGIα co-interaction. A and B, distribution of RhoA in cytosolic and membrane fractions prepared from Swiss 3T3 fibroblast cells 48 h after transient transfection with catalytically inactive (dead) PKGIα (KI). Cells were treated for 30 min with control or LPA (50 μm) in the absence or presence of 8-Br-cGMP (100 μm). B, summary densitometry data. C and D, agarose affinity assay: PKGIα or kinase-dead Iα and RhoA. FLAG-RhoA was transiently co-expressed with either PKGIα or kinase-dead PKGIα in COS-1 cells, followed by isolation on cGMP-agarose for 2 h at 4 ºC. RhoA and PKGIα complexes were detected by immunoblotting (IB) with anti-RhoA or anti-PKGIcom antibody, respectively. D, summary densitometry data. Data are representative of three independent experiments. * p < 0.05 versus all other groups. NS, not significant. Agarose, agarose beads alone; cGMP-agarose, cGMP-conjugated agarose beads. ADU, arbitrary densitometric units.