Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases

Abstract

The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.

FIG. 1.

Subcellular localization of CrkL in fibroblasts that express active Src. (A) CRKL-GFP or CRKL(K39)-GFP was transiently expressed in wild-type, Csk^−/−, Src^−/−, Csk^−/−, and Fyn^−/− Csk^−/− MEFs as indicated. Localization of the fusion protein was determined by GFP fluorescence. Antipaxillin was used as a marker for focal adhesions. (B) An activated mouse Src (SrcY529F) was permanently expressed in Crkas^−/− cells and their control cells with reintroduced Cas (Crkas^−/− + Cas) by pBABE puro retroviral vector. Subcellular localization of endogenous CrkL and vinculin was determined by immunofluorescence staining. F-actin was stained by phalloidin. Arrows indicate aggregation of F-actin, CrkL, and vinculin in podosome-like adhesion structures (overlap of the three colors show large whitish dots).

FIG. 2.

Subcellular localization of CrkL in migrating cells. (A to C) Subcellular localization of endogenous CrkL, paxillin, and F-actin. NIH 3T3 cells were plated on glass coverslips coated with fibronectin. Photographs of migrating cells were taken 3 h after generation of the cell-free zone. (A) The upper row shows quiescent NIH 3T3 cells; the lower row shows migrating NIH 3T3 cells. (B) The panel shows focal adhesion localization of CrkL in a large subset of migrating NIH 3T3 cells. (C) A higher magnification of the part of the cell highlighted by a white rectangle in panel B. The images are merged for three components: F-actin, CrkL, and paxillin. The panel at the right shows enlarged focal adhesion structures, where whiteness indicates overlap among F-actin, CrkL, and paxillin.

FIG. 3.

Subcellular targeting of CRKL to focal adhesions rescues motility defects of SYF cells. (A) The panel illustrates the structures of fusion proteins used in this study. (B) Immunoblot analysis for endogenous CrkL and FA-CRKL proteins expressed in cells used in panel C. (C) Modified Boyden chamber haptotaxis assay. The number of cells (cells migrated/field) that had traversed to reach the lower surface coated with fibronectin through pores in the insert that separated two chambers was determined 3 h after plating cells into the upper chamber. Error bars indicate the standard deviation of the sample data obtained in four individual microscopic fields under a 40× objective lens. Similar results were obtained in three independent experiments.

FIG. 4.

CrkL-deficient cells display haptotaxis defects. (A) Immunoblot analysis for CrkL and Crk-II in MEFs used for panel B. (B) Modified Boyden chamber migration assays. Haptotaxis toward fibronectin was determined as shown in Fig. 3C. Chemotaxis toward serum was determined in an assay similar to the haptotaxis assay except that both sides of the insert were coated with fibronectin with or without fetal bovine serum (10%) (FBS + or −, respectively) included in the lower chamber. Error bars indicate the standard deviation of the sample data obtained in four independent microscopic fields under a 40× objective lens. Three independent experiments yielded similar results.

FIG. 5.

FA-CRKL induced cytoskeletal reorganization. (A) Expression of FA-CRKL in NIH 3T3 cells induced multiple membrane protrusions decorated by lamellipodia (arrows). The part highlighted by a white rectangle was enlarged and shown at right. Blue arrowheads indicate FA-CRKL localized to focal adhesions. Green arrowheads indicate FA-CRKL localization at the membrane cortex where F-actin (red arrowheads) also colocalized. Merged color images use red and green to indicate localization of F-actin and FA-CRKL, respectively. (B) Cytoskeletal reorganization induced by transient expression of mutants of FA-CRKL. Localization of F-actin (red) and GFP (green) signals for fusion proteins is shown. Arrowheads indicate membrane protrusions decorated by lamellipodia. (C) Subcellular targeting of CRKL to the outer membrane of mitochondria. Localization of F-actin (red), CRKL-GFP-mito (green), and paxillin (blue) is shown. (D) The incidence of lamellipodial formation was counted after transient expression of each transgene indicated. Percent values indicate the number of NIH 3T3 cells with more than two membrane protrusions decorated by lamellipodia per total transfected (GFP-positive) cells. To assist observation of lamellipodia, cells were stained with phalloidin. Error bars indicate the standard deviation of the sample data collected in three independent sets of samples, each of which was scored with at least 60 cells in random fields under a 40× objective lens. (E) The number of protrusions containing lamellipodia per cell (x axis) was counted in each cell and plotted for occurrence (y axis) in NIH 3T3 cells transiently expressing FA-CRKL, FA-CRKL(ΔSH3n), or FA-GFP. For example, four cells had seven lamellipodium-containing protrusions in the FA-CRKL group. A total of 65 cells was counted for each group. Transient expression shown in panels B to E was performed at a dose of 0.1 μg/well of the expression plasmid, except that the experiments for panel D were performed at a dose of 0.3 μg/well.

FIG. 6.

Activation of small G proteins by FA-CRKL. (A) Immunoblot assay for expression levels of FA-CRKL compared to those of endogenous CRKL in NIH 3T3 cells expressing empty vector (CTR; lane 1), FA-CRKL, or FA-CRKL(ΔSH3n). (B) Pulldown assays for GTP-loaded Rac1, Cdc42, and Ras. Overall levels of these small G proteins were determined in parallel by using total cell lysates from NIH 3T3 cells expressing empty vector (CTR; lane 1), FA-CRKL (lane 2), or FA-CRKL(ΔSH3n) (lane 3) (shown at left). GTP-loaded forms were pulled down as described in Materials and Methods, and their levels were determined by immunoblotting by using specific antibodies to these small G proteins (shown at right). Levels of the GTP-loaded form were compared to total levels (not discriminating among GTP- and GDP-bound forms) of the G protein determined in total cell lysates. Experiments were repeated three times and yielded similar results. (C) The effects of dominant-negative forms of small G proteins were evaluated by counting the number of cells that show multiple membrane protrusions induced by FA-CRKL. Increasing doses of expression plasmids for Rac1 N17, Cdc42 N17, RhoA N17, or Ras N17 were cotransfected into NIH 3T3 cells coexpressing FA-CRKL at a fixed dose of 0.1 μg/well. Cells were stained with phalloidin to aid scoring samples. Values indicate the percentage of cells with lamellipodia among the total number of GFP-positive transfected cells. Error bars indicate the standard deviation of the sample values obtained in three independent sets of samples. (D) Subcellular localization of Dock1 in NIH 3T3 cells that express either FA-CRKL or FA-CRKL(ΔSH3n). Localization of fusion proteins was determined by GFP fluorescence. A part of the FA-CRKL-expressing cell highlighted by the white square was enlarged to show details of colocalization of FA-CRKL with Dock1.