Regulation of the Rho family small GTPase Wrch-1/RhoU by C-terminal tyrosine phosphorylation requires Src

Abstract

Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

FIG. 1.

Wrch-1 rapidly relocalizes upon serum stimulation. H1299 NSCLC cells transiently transfected to express GFP-Wrch-1 were grown in complete culture medium, then serum starved overnight, or first serum starved and then serum stimulated. Prior to serum stimulation, the treated cells were incubated for 1 h with AlexaFluor 647-labeled transferrin to mark endosomal compartments. After 15 min of serum stimulation, cells were fixed and then subjected to confocal microscopy for visualization of GFP-Wrch-1 (green) or transferrin (red). Bars, 20 μm.

FIG. 2.

Wrch-1 is tyrosine phosphorylated on Y254 in response to serum, and this phosphorylation is required for serum-stimulated relocalization. (A) Nonphosphorylatable Wrch-1(Y254F) is resistant to serum-stimulated relocalization. H1299 cells expressing either GFP-Wrch-1 or GFP-Wrch-1(Y254F) were grown, treated, and evaluated as described for Fig. 1. Bars, 20 μm. (B) Serum-stimulated tyrosine phosphorylation of Y254. H1299 cell lysates from cells expressing empty vector (VO), HA-Wrch-1, or HA-Wrch-1(Y254F) were incubated with anti-HA antibody. Immunoprecipitated (IP) Wrch-1 was detected by immunoblotting (IB) with anti-HA, and phosphotyrosine (p-Tyr) on Wrch-1 was detected by immunoblotting with antiphosphotyrosine antibody. The bands above and below the Wrch-1 band represent immunoglobulin heavy chain and light chains, respectively. Apparent molecular masses are shown (in kilodaltons).

FIG. 3.

The C-terminal 19 amino acids of Wrch-1 are sufficient for Wrch-1 to become tyrosine phosphorylated and to be relocalized in response to serum. (A) Schematic of the 19-aa and 9-aa tails. Shown are the GFP-tagged sequences extended with 9 or 19 amino acids of the C terminus of Wrch-1. Y254 is the only tyrosine residue present in each fusion protein. (B) Serum-stimulated tyrosine phosphorylation of the C-terminal 19 but not 9 amino acids of Wrch-1. H1299 cell lysates expressing either empty vector (GFP), GFP-Wrch-1 (FL), GFP fused to the 19-aa tail of Wrch-1, or GFP fused to the 9-aa tail ofWrch-1 were incubated with anti-GFP antibody to immunoprecipitate Wrch-1. Immunoprecipitated (IP) Wrch-1 was then detected by immunoblotting (IB) with anti-GFP antibody, and phosphotyrosine Wrch-1 was detected by immunoblotting with anti-phosphotyrosine antibody. (C) The C-terminal 19 amino acids of Wrch-1 are sufficient for serum-stimulated relocalization. H1299 cells as in panel B were grown, treated, and evaluated as described for Fig. 1. Bars, 20 μm.

FIG. 4.

Src activity is required in vivo for tyrosine phosphorylation of Wrch-1. (A) The Src family tyrosine kinase inhibitor SU6656 prevents Wrch-1 tyrosine phosphorylation in response to serum stimulation. H1299 cells expressing HA-Wrch-1 were serum starved overnight, treated with 5 μM SU6656 for 1 h, and serum stimulated for 5 min. Lysates were subjected to immunoprecipitation (IP) with anti-HA to retrieve HA-Wrch-1, followed by immunoblotting (IB) for Wrch-1 (anti-HA) or phosphotyrosine (anti-p-Tyr). Bands above and below the Wrch-1 band represent the Ig heavy chain and light chain, respectively. (B) Endogenous Src is required for serum-stimulated Wrch-1 tyrosine phosphorylation. SYF^−/− MEFs (MEFs lacking Src, Yes, and Fyn) and YF^−/− MEFs (MEFs retaining Src but lacking Yes and Fyn) expressing either HA-Wrch-1 or nonphosphorylatable HA-Wrch-1(Y254F) were starved overnight and then serum stimulated for 5 min. The resulting cell lysates were probed for phosphotyrosine on Wrch-1 as described for panel A. (C) Src kinase activity is required for tyrosine phosphorylation of Wrch-1. H1299 cells were cotransfected with empty vector, HA-Wrch-1, or nonphosphorylatable HA-Wrch-1(Y254F) along with either empty vector, kinase-active Src (Src Y528F), or kinase-deficient Src (Src K297R). Phosphotyrosine on Wrch-1 was detected as shown in panel A. (D) Src directly phosphorylates Wrch-1 in vitro. Purified recombinant GST-Wrch-1 protein was incubated with purified recombinant Src tyrosine kinase protein and [³²P]ATP. Total protein was detected by Coomassie blue staining, and [³²P]ATP incorporation was detected by autoradiography. (E) Inhibition of Src kinase with SU6656 blocks serum-stimulated relocalization of Wrch-1. H1299 cells expressing GFP-Wrch-1 or nonphosphorylatable GFP-Wrch-1(Y254F) were grown in complete culture medium (basal) and then either serum starved overnight (serum starved) or first serum starved and then serum stimulated for 15 min, with or without 1 h of pretreatment with 5 μM SU6656. Cells were visualized as for Fig. 1 for Wrch-1 (green) or transferrin (red). Bars, 20 μm.

FIG. 5.

Phosphorylation at Y254 negatively regulates Wrch-1-mediated biological functions. (A) Phosphorylatable tyrosine residue Y254 negatively regulates anchorage-independent growth. MDCKII cell lines were generated to stably express Wrch-1 proteins as indicated. Equivalent expression was confirmed by immunoblotting with anti-HA. β-Actin was used as a loading control. Cells were seeded into soft agar for anchorage-independent colony formation and grown for 14 days, then stained with MTT. Small colonies (6 to 15 cell diameters across) and large colonies (>15 cell diameters across) were quantified. Bar graphs indicate three independent experiments carried out in triplicate. One-way ANOVA and Tukey's post hoc tests were used to determine the significance of differences between numbers of colonies arising from cells expressing Wrch-1 with or without the Y254F mutation, in either the WT or constitutively activated (107L) backgrounds. **, P < 0.001. (B) Phosphorylatable tyrosine residue Y254 negatively regulates epithelial cell morphogenesis. MDCKII cells expressing Wrch-1 as in panel A were seeded into 3D collagen matrices and allowed to form cysts for 10 days. Cyst structures were evaluated and quantified according to whether they contained one lumen (normal; single lumen), more than one lumenal area (multilumen), or no lumen at all. Bars are the averages of three independent experiments for each cell line, with standard deviations. The significance of differences between numbers of normal cysts with single lumens in cells of different backgrounds was determined as described for panel A. *, P < 0.01.

FIG. 6.

Wrch-1 is GTP bound and active and recruits its effector PAK1 at the plasma membrane but not at endosomes. (A) Serum stimulation decreases recruitment of GFP-PAK-PBD to the plasma membrane by WT but not phospho-deficient Y254F Wrch-1. H1299 cells coexpressing GFP-PAK-PBD along with vector or Wrch-1 proteins were grown, treated, and evaluated as described for Fig. 1, except images were taken at multiple time points as shown. GFP-PAK-PBD signal is green and HA-Wrch-1 is red. Overlapping localization (merge; yellow) demonstrates recruitment of GFP-PAK-PBD by Wrch-1. Bars, 20 μm. (B) Quantitation of GFP-PAK-PBD subcellular distribution before and after serum stimulation. For each condition depicted with representative images in panel A, 50 cells from each of three independent experiments were evaluated for their subcellular distribution of GFP-PAK-PBD and quantified according to whether GFP-PAK-PBD localized primarily to the cytosol, the plasma membrane, or endosomal compartments. Results are presented graphically as the percentage of total cells counted in which the primary localization of GFP-PAK-PBD was identified as the specific compartment indicated. Bars represent the averages of three independent experiments for each cell line, with standard deviations.

FIG. 6.

Wrch-1 is GTP bound and active and recruits its effector PAK1 at the plasma membrane but not at endosomes. (A) Serum stimulation decreases recruitment of GFP-PAK-PBD to the plasma membrane by WT but not phospho-deficient Y254F Wrch-1. H1299 cells coexpressing GFP-PAK-PBD along with vector or Wrch-1 proteins were grown, treated, and evaluated as described for Fig. 1, except images were taken at multiple time points as shown. GFP-PAK-PBD signal is green and HA-Wrch-1 is red. Overlapping localization (merge; yellow) demonstrates recruitment of GFP-PAK-PBD by Wrch-1. Bars, 20 μm. (B) Quantitation of GFP-PAK-PBD subcellular distribution before and after serum stimulation. For each condition depicted with representative images in panel A, 50 cells from each of three independent experiments were evaluated for their subcellular distribution of GFP-PAK-PBD and quantified according to whether GFP-PAK-PBD localized primarily to the cytosol, the plasma membrane, or endosomal compartments. Results are presented graphically as the percentage of total cells counted in which the primary localization of GFP-PAK-PBD was identified as the specific compartment indicated. Bars represent the averages of three independent experiments for each cell line, with standard deviations.

FIG. 7.

Serum stimulation decreases active Wrch-1-GTP. To pull down active Wrch-1-GTP with GST-PAK-PBD, H1299 cells were transiently transfected with pCGN vector or vector encoding HA-tagged Wrch-1 proteins, then treated as for Fig. 6. Resulting cell lysates were incubated with GST-PAK fusion protein attached to glutathione-agarose beads. The Wrch-1/GST-PAK-PBD complex was collected by centrifugation, washed, eluted from the beads, and resolved by SDS-PAGE. Wrch-1 was detected by immunoblotting with anti-HA antibody. (Upper panels) Pull down followed by immunoblotting (GTP-Wrch-1); (lower panels) immunoblot of input total Wrch-1 available for pull down in the samples.

FIG. 8.

Serum-stimulated Wrch-1 tyrosine phosphorylation results in decreased Wrch-1 effector activation. (A) Serum stimulation results in decreased autophosphorylation of PAK1. Cells treated as described for Fig. 7 were probed for total PAK with an anti-PAK1/2/3 antibody or for active phospho-PAK with an anti-phospho(Thr423)-PAK1/phospho(Thr402)-Pak2 antibody. (B) Serum stimulation results in decreased Pyk2 autophosphorylation. Cells treated as for panel A were probed for total Pyk2 with anti-Pyk2 antibody and for phospho-Pyk2 with anti-phospho(Tyr402)-Pyk2 antibody. Densitometry was performed using ImageJ software. Phosphoprotein was normalized to total protein in each lane, and the percent phosphorylation shown represents phosphorylation compared to no (0 min) serum stimulation.