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srGAP2 arginine methylation regulates cell migration and cell spreading through promoting dimerization - PubMed

  • ️Fri Jan 01 2010

srGAP2 arginine methylation regulates cell migration and cell spreading through promoting dimerization

Shaoshi Guo et al. J Biol Chem. 2010.

Abstract

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225-538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.

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Figures

FIGURE 1.
FIGURE 1.

srGAP2 influences cell migration. A, Western blot with anti-srGAP2 and anti-actin antibodies, 100 μg of whole cell lysates from HCT116 cells transfected with shRNA. U1 and U2 are two shRNAs of srGAP2 and siLuc as control. B, 100 μg of whole cell lysates from HCT116 cells transfected with either GFP-siLuc (as control) or GFP-U1 probed with anti-srGAP2 and anti-actin antibodies. C, colony formation assay: HCT116 cells transfected with shRNAs as indicated. D, Western blot showing the srGAP2 expression levels of HCT116 stable cell lines. LucD2 was stably expressing siLuc, U1A5 or U2A4, U2B1 and U2B3 represented the clone number stably expressing srGAP2-shRNA U1 or U2. Actin shows equal loading. E, growth curves of control cells (LucA1, LucA8, and LucD2) and srGAP2-depleted cells in D by MTS analysis. F, wound healing assay. The confluent monolayers of LucD2 (control), U1A5, and U2B1 cells were scratched to remove cells, and images of the cells were collected as the 0′ hour time point (left panels). The cells were then cultured for 48 h, and captured at the same point of 0′ hour time points as the 48′ hour time point (panels). Scale bar, 50 μm. G, quantitative analysis of cell migration showing the areas of wound closure (%) compared with initial wound areas (0 h) using NIH image J analysis. Data are mean ± S.E. (n = 3; **, p < 0.001; *, p < 0.05; Student's t test).

FIGURE 2.
FIGURE 2.

srGAP2 localizes to sites of membrane protrusion and is required for cell morphology. A, HCT116 cells at 24 h after seeding on the coverslip were double labeled with mouse anti-srGAP2 antibody, followed by anti-mouse FITC-conjugated secondary antibody and TRITC 545-phalloidin. Bars, 10 μm. The lower figures are the high-magnification from the upper selected inset, showing that srGAP2 localized to leading edge of F-actin at the cell membrane protrusion. B, graphs correspond to intensities in arbitrary units of the green (srGAP2) and red (F-actin) labeling for each pixel of the arrow drawn through the axis in A. C, HeLa cells transfected with GFP or GFP-srGAP2. The arrow indicated filopodia-like protrusion. Bars, 10 μm. D and E, srGAP2 regulates the cell morphology. Graphs taken at 33 h after seeding, LucD2 (control) showed well-pronounced protrusions and spread well, U1A5 (srGAP2 depleted) became rounded. DNA was stained with DAPI (blue) and visualized by DIC (D). Cells were stained with TRITC 545-phalloidin to visualize F-actin (red) (E). Bars, 10 μm.

FIGURE 3.
FIGURE 3.

srGAP2 is required for cell spreading. A, cells were stained at indicated times with TRITC 545-phalloidin to visualize F-actin (red) and DAPI to indicate nuclei (blue). Bars, 10 μm. B, cell spreading of either LucD2 or U1A5 at each time point was counted using the cell spreading assay. Data are mean ± S.E. (n = 3; *, p < 0.05, Student's t test). C, rescuing assay: U1A5 transfected with GFP or srGAP2-R-GFP and stained with TRITC 545-phalloidin to visualize F-actin distribution 22 h after transfection. Bars, 5 μm. D, cell spreading situation of either U1A5 or U1A5 transfected with GFP or srGAP2-R-GFP at each indicated time point (n1/n2: n1 means the length of time after plating the cells, and n2 means the length of time after transfection) was counted using the cell spreading assay; see “Experimental Procedures” for details. Data are mean ± S.E. (n = 3; *, p < 0.05; Student's t test.). E, time-lapse confocal microscopy analysis showing the slowness of extension and spreading after mitosis. Living U2OS cells transfected with the GFP-U1 plasmid was indicated with an arrowhead, and the untransfected control cell shown with an arrow. Living cells were imaged at 8-min intervals.

FIGURE 4.
FIGURE 4.

srGAP2 interacts with PRMT5. A, endogenous PRMT5 and srGAP2 interact with each other. HEK293 cell lysates were subjected to immunoprecipitation with anti-srGAP2 or preimmunoserum (Pre-I), and anti-PRMT5 or IgG antibodies as indicated. The immunocomplexes were separately and probed for PRMT5 and srGAP2. B, diagrams of different fragment of srGAP2. C, pull-down assay to test interaction of Flag-tagged PRMT5 with GST-tagged N (1–538 aa)/C (484–1068 aa)-terminal srGAP2 and N (1–373aa)/C (367–743) -terminal Reps1. Western blot analysis was performed with GST and Flag antibodies. D, pull-down assay to test the interaction of Flag-tagged PRMT5 with different fragments of GST-tagged srGAP2-N-terminal. Each protein is indicated with an arrow. Western blot analysis was performed with GST and Flag antibodies.

FIGURE 5.
FIGURE 5.

PRMT5 methylates srGAP2 in vitro. A, methylation assay in vitro. 2–5 μg of GST, GST-tagged srGAP2-N, N1, N2, and N3, and His-tagged C, C2, and C3 were purified from E. coli, and incubated with 1 μg of Flag-PRMT5, purified from CHO cells. Incorporation of 3H-labeled methyl groups (from the SAM donor) was visualized by autoradiography. Each protein is indicated with an arrow. B, 30.4 kDa methylated polypeptide band (srGAP2-C3) in Fig. 5A was fragmented and analyzed by mass spectrometry, and the result indicated the molecular weight of srGAP2 fragments and assignment of the methylated arginine. Note that the mass shifts reflect the tritium molecular weight. * represents 3H-labeled -CH3. C, arginine residues in srGAP2 C and C3 fragments were mutated to alanine (R927A). 2–5 μg of His-tagged C, C2, C3, C4, and the R927A mutants in C (Cm) and C3 (C3m) were purified from E. coli, and the methylation assay as in A. Each protein is indicated with an arrow. D, amino acid sequence alignment of human Arg-927 site of srGAP2, marked with *, in some eukaryotic species was performed using DNAMAN version 4.0 software. Conserved residues are highlighted with the following color type: black (=100%) and gray (≥75%).

FIGURE 6.
FIGURE 6.

The functional analysis of srGAP2 arginine methylation. A, HCT116 cells cotransfected with shRNA and GFP-tagged srGAP2 as indicated, and whole cell extracts were probed with antibodies against GFP or actin (loading control). As shown, srGAP2-R-GFP and srGAP2-dm-GFP were resistant to U1 (srGAP2 shRNA). B and C, U1A5 transfected with srGAP2-R-GFP (B) or srGAP2-dm-GFP (C) were stained with TRITC 545-phalloidin to visualize F-actin distribution 22 h after transfection. The right figures are the high-magnification from the left selected inset, showing the distribution srGAP2 and F-actin at the cell membrane protrusion. Bars, 5 μm. D, cell spreading situation of either srGAP2-depleted HCT116 cells (U1A5) transfected with srGAP2-R-GFP or srGAP2-dm-GFP at each indicated time point (n1/n2: n1 means the length of time after plating the cells, and n2 means the length of time after transfection) was counted using the cell spreading assay; see “Experimental Procedures” for details. Data are mean ± S.E. (n = 3; *, p < 0.05, Student's t test). E and F, graphs correspond to intensities in arbitrary units of the green and red labels for each pixel of the arrow drawn through the axis. Graph E is from the arrow in B, and graph F is from the arrow in C.

FIGURE 7.
FIGURE 7.

srGAP2 arginine methylation promotes the dimerization of srGAP2. A, co-immunoprecipitation was performed in HEK293 cells cotransfected with two plasmids, as indicated. IP: antibodies used in immunoprecipitation; Western blot with the anti-GFP antibody first, then blotted with anti-HA antibody after stripping away GFP antibody. B, GST-PBD pull-down assay. GST-PBD was performed to pull down the associated proteins from cell lysates, and followed by Western blot using anti-srGAP2 and Rac1 antibodies. GST-PBD was indicated with Ponceau S staining. Lanes from left to right: LucD2, U1A5, U1A5 transfected with GFP or srGAP2-R-GFP and/or srGAP2-m-GFP. C, co-immunoprecipitation was performed in HEK293 cells cotransfected with the indicated plasmids. IP: antibodies used in immunoprecipitation. Western blot with the anti-GFP and -HA antibodies. D, GFP-tagged srGAP2 binding relative to HA-tagged srGAP2, normalized from C, lanes 3–5, analyzed using the NIH Image J software. WT-WT: srGAP2-GFP pulled by HA-srGAP2; MT-MT: srGAP2-m-GFP pulled by HA-srGAP2-m; MT-WT: srGAP2-m-GFP pulled by HA-srGAP2.

FIGURE 8.
FIGURE 8.

Methylation of srGAP2 is required for filopodia protrusions and cell spreading. Hypothetical model of the molecular mechanisms illustrating that methylation of srGAP2 promotes the dimerization of srGAP2, plasma membrane protrusions, and cell spreading. See text for details.

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