The Src substrate SKAP2 regulates actin assembly by interacting with WAVE2 and cortactin proteins

Abstract

In our attempt to screen for substrates of Src family kinases in glioblastoma, Src kinase-associated phosphoprotein 2 (SKAP2) was identified. Although SKAP2 has been suggested to be associated with integrin-mediated adhesion of hematopoietic cells, little is known about its molecular function and the effects in other types of cells and tumors. Here, we demonstrate that SKAP2 physically associates with actin assembly factors WAVE2 and cortactin and inhibits their interaction. Cortactin is required for the membrane localization of WAVE2, and SKAP2 suppresses actin polymerization mediated by WAVE2 and cortactin in vitro. Knockdown of SKAP2 in NIH3T3 accelerated cell migration and enhanced translocation of WAVE2 to the cell membrane, and those effects of SKAP2 depend on the binding activity of SKAP2 to WAVE2. Furthermore, reduction of SKAP2 in the glioblastoma promoted tumor invasion both in ex vivo organotypic rat brain slices and immune-deficient mouse brains. These results suggest that SKAP2 negatively regulates cell migration and tumor invasion in fibroblasts and glioblastoma cells by suppressing actin assembly induced by the WAVE2-cortactin complex, indicating that SKAP2 may be a novel candidate for the suppressor of tumor progression.

FIGURE 1.

SKAP2 localizes at the cell membrane and attenuates cell migration and invasion. A and B, NIH3T3 cells were immunostained using anti-SKAP2 (green) and anti-paxillin (A, red) antibodies or phalloidin (B, red). Bottom panels in B are enlarged images of the upper panels. Bar, 10 μm. C, top, structure of mouse SKAP2. SKAP2 consists of a coiled-coil domain, a PH domain, and an SH3 domain. The W336K SKAP2 mutation is represented. Bottom, SKAP2 was stably knocked down (KD) in NIH3T3 and U87MG cells using miRNA, and wild-type or W336K mutant SKAP2 was stably added back to the SKAP-KD cells. The expression level of SKAP2 was examined by immunoblotting (IB) using an anti-SKAP2 antibody. aa, amino acids. D, motility of NIH3T3 and U87MG cell lines was examined by the transwell assay. Top, representative pictures of migrated cells on the bottom surface of the transwell membrane. Bottom, number of migrated cells was counted. E, top, invasion of NIH3T3 cell lines into a matrix containing type I-collagen and Matrigel. The indicated cells were labeled with DiI (red), overlaid on top of the gel in the transwells, and incubated for 72 h. The gels were fixed and sliced and observed using confocal microscopy. Representative pictures are shown. Bar, 100 μm. *, position of the gel. Bottom, invading chains of cells were quantified by measuring the area of protrusion into the gel from the arc of cell clumps. D, bottom, and E, bottom, the amount of wild-type cells was defined as 1. The results from three independent experiments are shown as the means ± S.D. *, p < 0.01.

FIGURE 2.

SKAP2 overexpression inhibits cell migration and invasion. A, SKAP2 was stably overexpressed in U87F4 and C6 cells using lentivirus. The expression level of SKAP2 was examined by immunoblotting (IB) using an anti-SKAP2 antibody. B, motility of U87F4 and C6 was examined by the transwell assay as in Fig. 1D. C, invasion of U87F4 and C6 into a matrix containing type I collagen and Matrigel was analyzed as in Fig. 1E. Bar, 100 μm. *, position of the gel.

FIGURE 3.

SKAP2 physically interacts with WAVE2. A, top, structure of human WAVE2. WHD, WAVE homology domain; VCA, verprolin homology, cofilin homology and acidic region; aa, amino acids. Bottom, COS-1 cells were transfected with wild-type or the W336K SKAP2 mutant tagged with FLAG at the amino terminus (FLAG-SKAP2) and carboxyl-terminally HA-tagged WAVE2 (WAVE2-HA). FLAG-SKAP2 was immunoprecipitated (IP) using an anti-FLAG antibody. WAVE2 and SKAP2 were detected using anti-HA and anti-SKAP2 antibodies, respectively. The arrow indicates WAVE2-HA. B, COS-1 cells were transfected as described in A. WAVE2-HA was immunoprecipitated using an anti-HA antibody, and SKAP2 and WAVE2 were detected. C, endogenous WAVE2 in NIH3T3 cells was immunoprecipitated using an anti-WAVE2 antibody or the control normal rabbit IgG. SKAP2 and WAVE2 were detected using anti-SKAP2 and anti-WAVE2 antibodies, respectively. The asterisk represents nonspecific bands. IB, immunoblot. D, top, diagram of the recombinant GST fusion SKAP2 fragments used in this study. Middle, protein extract of COS-1 transfected with WAVE2-HA was pulled down by GST alone, the GST-PH domain, or the SH3 domain of SKAP2, and immunoblotted using an anti-HA antibody. Bottom, GST fusion proteins bound to glutathione-Sepharose beads were electrophoresed and analyzed using an anti-GST antibody. E, COS-1 cells were transfected with FLAG-SKAP2, WAVE2-HA, and Fyn tyrosine kinase and analyzed as in A. The phosphorylation of SKAP2 in FLAG IP fractions was detected using an anti-phosphotyrosine antibody.

FIGURE 4.

SKAP2 colocalizes with WAVE2 and affects its localization. A, SKAP2-KD NIH3T3 cells reconstituted with SKAP2-HA were cultured on fibronectin-coated coverslips for 3 h and immunostained using anti-HA (magenta) and anti-WAVE2 (green) antibodies. Bar, 10 μm. B, NIH3T3 cell lines were cultured on fibronectin for 30 min and immunostained using anti-HA (magenta) and anti-WAVE2 (green) antibodies and phalloidin (red). W336K, W336K SKAP2 mutant. Bar, 10 μm. C, number of cells bearing membrane-localized WAVE2 was counted in a total of 50 cells for each in A and B. D, wild-type and SKAP2-KD NIH3T3 cells were subjected to subcellular fractionation as described under “Experimental Procedures.” WAVE2 in each fraction was analyzed using an anti-WAVE2 antibody. ERK (MAPK) and CXCR4 were analyzed using anti-ERK and anti-CXCR4 antibodies as controls of proteins in cytoplasm and cell membrane, respectively. IB, immunoblot.

FIGURE 5.

SKAP2 interacts with cortactin. A, top, structure of human cortactin. NTA, amino-terminal acidic region; aa, amino acids. Bottom, NIH3T3 cells were immunostained using anti-SKAP2 (green) and anti-cortactin (magenta) antibodies and phalloidin (red). Bar, 10 μm. B, COS-1 cells were transfected with cortactin-HA with or without FLAG-SKAP2 or W336K mutant. FLAG-SKAP2 was immunoprecipitated using an anti-FLAG antibody. Cortactin and SKAP2 were detected using anti-HA and anti-SKAP2 antibodies, respectively. C, COS-1 cells were transfected as indicated. Cortactin-HA was immunoprecipitated (IP) using an anti-HA antibody, and SKAP2 was detected. D, endogenous SKAP2 in NIH3T3 cells was immunoprecipitated using an anti-SKAP2 antibody. Cortactin and SKAP2 were detected using anti-cortactin and anti-SKAP2 antibodies, respectively. E, top, protein extracts from COS-1 cells transfected with cortactin-HA were pulled down by GST alone, the GST-PH domain, or the SH3 domain of SKAP2 and immunoblotted (IB) with an anti-HA antibody. Bottom, immunoblot of GST fusion proteins was shown.

FIGURE 6.

Cortactin recruits WAVE2 to the cell membrane and SKAP2 inhibits their interaction. A, COS-1 cells were cotransfected with EGFP-WAVE2 and cortactin-HA. Cortactin-HA was immunoprecipitated (IP) using an anti-HA antibody. WAVE2 and cortactin were detected using anti-EGFP and anti-HA antibodies, respectively. B, endogenous WAVE2 in NIH3T3 cells were immunoprecipitated using an anti-WAVE2 antibody. Cortactin and WAVE2 were detected using anti-cortactin and anti-WAVE2 antibodies, respectively. C, left, control, cortactin-KD and human cortactin added-back NIH3T3 cells were immunostained using anti-cortactin (magenta) and anti-WAVE2 (green) antibodies. Bar, 10 μm. Top right, cortactin was transiently knocked down in NIH3T3 cells using siRNA, and human cortactin was transiently added back. The expression level of cortactin was examined by immunoblotting (IB) with an anti-cortactin antibody. Bottom right, percentages of signal intensities of WAVE2 at the cell periphery compared with those in the whole cell were quantified using ImageJ. D, FLAG-SKAP2 and Fyn in addition to cortactin-HA and EGFP-WAVE2 were expressed in COS-1 cells, and the interaction between WAVE2 and cortactin was analyzed (top panels). To confirm the phosphorylation of SKAP2 by Fyn, SKAP2 was immunoprecipitated using an anti-FLAG antibody, and detected by anti-FLAG and anti-phosphotyrosine antibodies (bottom panels).

FIGURE 7.

SKAP2 directly inhibits actin polymerization induced by the WAVE2-cortactin complex. A, top, diagram of the actin polymerization assay. Rhodamine-conjugated monomeric G-actin, WAVE2, and GST-SKAP2 were added to cortactin-coated protein G beads. HeLa cell lysates were also added to supply the Arp2/3 protein complex. After incubation, protein G beads were separated from the reaction mixture, washed, and subjected to immunoblotting (IB). Bottom, polymerized actin, which was produced in the complex of cortactin and WAVE2, was detected using anti-rhodamine and anti-actin antibodies. WAVE2, SKAP2, and cortactin were detected using anti-WAVE2, anti-SKAP2, and anti-EGFP antibodies, respectively. Sup, reaction mixture; beads, beads fraction after incubation. B, top, diagram of the actin polymerization assay using EGFP-WAVE2-coated beads. Bottom, polymerized actin was analyzed as A. A and B, signal intensities of rhodamine-actin in the beads fraction were quantified. Signal intensities of lanes with WAVE2 without SKAP2 were defined as 1. The results from the three independent experiments are shown as the means ± S.D.

FIGURE 8.

SKAP2 suppresses the invasion of glioblastoma in the mouse brain. A and B, invasion of glioblastoma in ex vivo organotypic rat brain slices. 3,3′-Dioctadecyloxacarbocyanine-labeled cells were spotted on the putamen of sliced rat brains and cultured for 72 h. The areas of the tumor were visualized through a fluorescent dissecting scope. A total of five brain explants for each group were examined, and representative images are shown. Bar, 500 μm. A, control, SKAP2-KD and SKAP2-added back U87MG. B, control, wild-type, or Y260F SKAP2-overexpressed U87F4 cells. C and D, glioblastomas were injected into nude mice brains. Mice were sacrificed 14 days later, and the brains were sliced and stained by H&E. Five mice in each group were examined, and representative images are shown. Bar, 100 μm. C, control (panel a), SKAP2-KD (panel b), and SKAP2 added back (panel c) U87MG cells. Panel a, a-1 to a-3, panel b, b-1 to b-3, and panel c, c-1 to c-3: high magnification fields of the boxed areas in panels a–c, respectively. Arrowheads indicate tumor cells invading along the capillary. D, control, wild-type, or Y260F SKAP2-overexpressed C6 cells.

FIGURE 9.

Diagram showing the possible mechanism of the regulation of actin polymerization and cell migration by SKAP2. Left, WAVE2 is recruited to cortactin at the cell membrane, polymerizes actin through Arp2/3 complex, and cell migration occurs. Middle, SKAP2 interferes with the association between WAVE2 and cortactin, and inhibits actin polymerization, which suppresses cell migration and tumor invasion. Right, SKAP2-phosphorylation by SFKs attenuates its inhibitory effect on WAVE2-cortactin interaction, cell migration and invasion.