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, 23 (12), 4219-29

RhoE Binds to ROCK I and Inhibits Downstream Signaling

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RhoE Binds to ROCK I and Inhibits Downstream Signaling

Kirsi Riento et al. Mol Cell Biol.

Abstract

RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.

Figures

FIG. 1.
FIG. 1.
RhoE interacts with ROCK I but not with PRK1 or mDia1 in vitro. (A) COS-7 cells were transiently transfected with expression vectors encoding myc-tagged ROCK I, myc-PRK1, FLAG-mDia1, or myc-citron kinase Δ1. Cells were lysed after 24 h, and cell lysates were incubated with GST, GST-RhoE, or GST-RhoA-V14 on glutathione beads. The precipitated complexes were analyzed by SDS-PAGE and immunoblotting. The amounts of GST fusion proteins were determined by Coomassie blue staining of the gel. (B) myc-ROCKΔ1 produced in COS-7 cells was immunoprecipitated with anti-myc antibody, washed with 0.5 M NaCl to remove ROCK-binding proteins, and eluted with myc-peptide. A sample of the eluate was resolved by SDS-PAGE and silver stained. The eluate was incubated with GST or GST-RhoE on glutathione beads, and the complexes were analyzed by SDS-PAGE and immunoblotting. WB, Western blot.
FIG. 2.
FIG. 2.
RhoE and RhoA bind to different domains of ROCK I. (A) Diagrammatic representation of ROCK I mutants used in this study. ROCK I is represented with its structural domains at the top. The region of ROCK I between the kinase domain and the pleckstrin homology (PH) domain is predicted to form a coiled-coil structure. Numbers indicate amino acid residues. CRD, cysteine-rich domain. (B) ROCKΔ3 binds RhoE but not RhoA. COS-7 cells were transiently transfected with expression vectors encoding myc-ROCKΔ1, -Δ3, or -Δ5, and the lysates were incubated with GST, GST-RhoE, or GST-RhoA-V14 on glutathione beads. Bound ROCK I was detected by an immunoblot analysis with anti-myc antibody. (C) The minimal region for RhoE binding is amino acids 1 to 420 in ROCK I. Plasmids encoding myc-tagged ROCK-CC, His-tagged ROCK M2-2, myc-ROCK R1, or myc-ROCK R2 were transfected into COS-7 cells. Cell lysates were incubated with GST, GST-RhoE, or GST-RhoA-V14 on glutathione beads. Bound proteins were detected by immunoblotting.
FIG. 3.
FIG. 3.
RhoE colocalizes with ROCK I on the Golgi complex. COS-7 cells were stained with polyclonal (A, D, and F) or monoclonal (B) anti-RhoE antibodies, anti-TGN38 antibody (E and H), or polyclonal anti-ROCK I antibody (G). Swiss 3T3 cells were stained with monoclonal RhoE antibody (C), anti-TGN38 antibody (K), or anti-FLAG antibody to stain exogenous FLAG-RhoE (I). Swiss 3T3 cells were microinjected with plasmid encoding FLAG-RhoE (I to K) and fixed after 5 h. Filamentous actin was stained with TRITC-phalloidin in panels A to E and J and is shown in blue in COS-7 cells to indicate the cell shape (A, B, D, and E) and in red in Swiss 3T3 cells (C and J). Bars, 20 μm (A, B, and F to H), 35 μm (C and I to K), and 15 μm (D and E). (L) Intracellular distribution of RhoE and ROCK I by cell fractionation and immunoblotting. RhoE and ROCK I were immunoblotted with polyclonal and monoclonal antibodies, respectively.
FIG. 4.
FIG. 4.
RhoE coimmunoprecipitates with ROCK I. (A) Lysates of COS-7 cells expressing myc-ROCKΔ1 and FLAG-RhoE or FLAG-RhoA-V14 were subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were separated by SDS-PAGE and analyzed by immunoblotting. (B) Lysates of Swiss 3T3 cells or COS-7 cells were subjected to immunoprecipitation with polyclonal anti-RhoE antibody or, as a control, with rabbit preimmune serum. Precipitates were immunoblotted with monoclonal RhoE and ROCK I antibodies. (C) COS-7 cells were cotransfected with expression vectors encoding myc-ROCK I wt or myc-ROCKΔ1, together with plasmids encoding FLAG-RhoE or FLAG-RhoA-V14. ROCK proteins were pulled down by GST-RhoA-V14 or GST-RhoE on glutathione beads. After washes the bound proteins were separated by SDS-PAGE and analyzed by immunoblotting. The cleaved form of ROCK I is indicated by an arrowhead on the immunoblot showing the levels of expressed proteins in cell lysates. IP, immunoprecipitation; WB, Western blot.
FIG. 5.
FIG. 5.
RhoE inhibits ROCK I- and RhoA-induced stress fiber assembly. Starved Swiss 3T3 cells were microinjected with plasmids encoding myc-ROCK I (25 μg/ml) (A and B), myc-ROCK I (25 μg/ml) and FLAG-RhoE (50 μg/ml) (C and D), myc-ROCKΔ1 (10 μg/ml) (E and F), myc-ROCKΔ1 (10 μg/ml) and FLAG-RhoE (100 μg/ml) (G and H), myc-RhoA (25 μg/ml) (I and J), or myc-RhoA (25 μg/ml) and FLAG-RhoE (100 μg/ml) (K and L). Cells were stained after 6 h of incubation for filamentous actin with rhodamine-conjugated phalloidin (A, C, E, G, I, and K). For the detection of myc-tagged proteins cells were stained with mouse 9E10 antibody (B, F, and J), and for the detection of FLAG-RhoE they were stained with mouse anti-FLAG antibody (D, H, and L). Bar, 30 μm. The percentages of injected cells that lack stress fibers as determined by using different FLAG-RhoE DNA concentrations are presented in panel M.
FIG. 6.
FIG. 6.
RhoE inhibits myosin phosphatase phosphorylation by ROCK I. COS-7 cells were transfected with plasmids encoding FLAG-RhoE, myc-ROCK I, and myc-ROCKΔ1. Equal amounts of proteins of the transfected-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting. The data are representative of three independent experiments.
FIG. 7.
FIG. 7.
PDGF induces changes in RhoE protein expression level. (A) Starved Swiss 3T3 cells were incubated with 25 ng of PDGF/ml for 1, 4, or 16 h, and the cells were stained for filamentous actin. Bar, 30 μm. (B) Equal amounts of lysates of starved and PDGF-treated Swiss 3T3 cells were separated by SDS-PAGE and immunoblotted with anti-ROCK I antibody or with polyclonal anti-RhoE antibody. The data are representative of three independent experiments.

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