Focal adhesions require catalytic activity of Src family kinases to mediate integrin-matrix adhesion

Abstract

Members of the Src family of tyrosine kinases function to phosphorylate focal adhesion (FA) proteins. To explore the overlapping functions of Src kinases, we have targeted Csk, a negative regulator of the Src family, to FA structures. Expression of FA-targeted Csk (FA-Csk) effectively reduced the active form (nonphosphorylated at the C-terminal regulatory tyrosine) of Src members in the cell. We found that fibroblasts expressing FA-Csk lost integrin-mediated adhesion. Activated Src (SrcY529F) as well as activation of putative Src signaling mediators (Fak, Cas, Crk/CrkL, C3G, and Rap1) blocked the effect of FA-Csk in a manner dependent on Rap1. SrcY529F also inhibited activated Ras-induced cell detachment but failed to rescue detachment caused by an activated mutant of Raf1 (Raf-BXB) that Rap1 cannot inhibit. Although normal spreading onto fibronectin was restored by the beta(1) integrin affinity-activating antibody TS2/16 in cells expressing FA-Csk or Raf-BXB, FAs were lost in these cells. On the other hand, Rap1 activation could restore FAs in cells expressing FA-Csk. Activation of the executioner caspase, caspase 3, is essential for many forms of apoptosis. While a caspase 3 inhibitor (Z-DEVD-FMK) inhibited cell detachment triggered by activation of caspase 8, this inhibitor had no effect on cell detachment caused by FA-Csk. Likewise, overexpression of an activated Akt made cells resistant to the effect of caspase 8 activation, but not to the effect of FA-Csk. It is therefore likely that the primary cause of cell rounding and detachment induced by FA-Csk involves dysfunction of FAs rather than caspase-mediated apoptosis that may result from possible loss of survival signals mediated by Src family kinases. We suggest that endogenous Src family kinases are essential for FAs through activation of Rap1 in fibroblasts.

FIG. 1.

Subcellular targeting of Csk to the FA complex. (A) Structures of Csk fusion proteins, Csk, Fak, and paxillin. PTK, protein tyrosine kinase domain; SH, Src homology domain; FAT, FA-targeting sequence; GFP, green fluorescent protein. The C-terminal half of paxillin contains four LIM domains. Kinase-inactive versions of Csk fusion proteins were also made with a point mutation that replaces the lysine residue (K222) at the ATP binding site with a methionine residue. (B) Subcellular localization of FA-GFP proteins (GFP-FAT and GFP-LIM) in NIH 3T3 cells was determined. Note that fusion proteins (green) are localized to FA structures at the distal tip of F-actin fibers (red). Another FA-GFP, GFP-Pxn, showed similar subcellular localization (not shown). Cells were fixed and stained with phalloidin 18 h after electroporation for transient expression of the transgene. (C) The ability of FA-targeted Csk to phosphorylate the C-terminal regulatory tyrosine of Src kinases was determined by Clone 28 immunoblot analysis. HEK 293 cells were transiently transfected by lipofection with 4 μg of kinase-active or -inactive CskGFP-FAT [FA-Csk(+) or FA-Csk(−), respectively], normal mouse Csk (Csk+), or empty vector (CTR) in a 100-mm-diameter tissue culture plate. Twenty-four hours later, cell lysates were made. Ten micrograms of total protein per lane was separated in an SDS-polyacrylamide gel and analyzed in immunoblots probed with Clone 28 (to detect Src kinases with the C-terminal tyrosine nonphosphorylated), MAb327 (to detect the total amount of Src expressed), or anti-Csk antibody (to determine the amount of transgene expression as well as endogenous Csk). All kinase-active FA-Csk constructs had similar effects on the phosphorylation status of the C-terminal tyrosine of Src kinases (not shown).

FIG. 2.

Loss of integrin-mediated adhesion caused by FA-Csk. (A) The morphologies of wild-type MEFs and NIH 3T3 cells expressing kinase-active or -inactive FA-Csk constructs were determined after transient expression of the transgene. Only one example of each construct with each cell type is shown. Phalloidin staining is shown in red. Green indicates GFP fluorescence in the transfected cell (arrows). (B) The numbers of GFP-positive NIH 3T3 cells remaining on fibronectin-coated coverslips were determined in 30 random fields under a 25× objective and expressed as percentages (± standard deviations [SD] in a triplicate experiment) of the control group electroporated with GFP alone. (C) The numbers of GFP-positive NIH 3T3 cells remaining attached were determined on an ECM protein or PLL (PLL). Cells were transfected by lipofection with CskGFP-FAT. FN, fibronectin; VN, vitronectin; Col, denatured collagen (gelatin). The values indicate the numbers of GFP-positive cells relative to those of the control group transfected with the kinase-inactive variant. (D) Morphology of NIH 3T3 cells expressing the kinase-active version of CskGFP-FAT on PLL-coated glass coverslips. Green and red indicate GFP fluorescence and F-actin, respectively. (E) NIH 3T3 cells were plated on glass coverslips coated with increasing concentrations of fibronectin prior to transient transfection with 0.1 μg of CskGFP-FAT construct per well by lipofection. The y axis shows the total number (mean ± SD in a triplicate experiment) of GFP-positive cells remaining on the tissue culture surface in each group. (F) NIH 3T3 cells were stained with anti-Csk antibody (α-Csk) after electroporation with kinase-inactive CskGFP-FAT in order to determine expression levels of the transgene compared to endogenous Csk. Green and white arrowheads indicate cells transfected and nontransfected, respectively. Levels of proteins identified by anti-Csk antibody were measured as described in Materials and Methods. (G) NIH 3T3 cells were plated on fibronectin-coated coverslips and stained with an anti-Fak antibody (α-Fak) after electroporation with kinase-negative CskGFP-FAT. This antibody detects endogenous Fak but not the fusion protein, as it recognizes the N terminus of Fak. Green or white arrowheads indicate endogenous Fak at FA structures in a transfected cell or in a nontransfected cell, respectively. In all experiments shown (panels A to G), cells were cultured in DMEM containing 10% calf serum for 18 h after transfection before fixation. All experiments were repeated at least three times.

FIG.3.

Src activation rescues the FA-Csk phenotype. (A) Kinase-active CskGFP-FAT was electroporated into wild-type MEFs or MEFs permanently expressing SrcY529F. Wild-type MEFs showed a spherical morphology (arrows) upon expression of FA-Csk(+), while SrcY529F-expressing MEFs were resistant. Cells were stained for F-actin (red). The panel next to the photomicrographs shows an immunoblot for Src for which total cell lysates were used. (B) FA-Csk plasmid (0.1 μg of kinase-active or -inactive CskGFP-FAT, indicated by closed or open circles, respectively) was cotransfected by lipofection into NIH 3T3 cells plated on fibronectin with increasing concentrations of activated Src (Src-Y529F), its kinase-inactive variant (Src-K297M/Y529F), or normal mouse Src. (C) Cotransfection of FA-Csk plasmid (0.1 μg of kinase-active or -inactive CskGFP-LIM; closed or open circles, respectively) with increasing concentrations of CD2Fak, CD2Fak-Y397F, CasCT, or CasCT-P642A was carried out as described above. (D) Increasing concentrations of the membrane-anchored p110α subunit of phosphoinositide 3" kinase (p110-CAAX; Myc-tagged) were cotransfected with 0.1 μg of FA-Csk plasmid per well in a 24-well plate (kinase-active or -inactive CskGFP-FAT, indicated by closed or open circles, respectively). The Western blot below panel E shows expression levels of p110α in NIH 3T3 cells transfected with 0, 0.3, or 3 μg of plasmid in a 60-mm-diameter plate (from left to right; approximately corresponding to 0, 0.03, and 0.3 μg of plasmid per well of a 24-well plate as performed in detachment assays, respectively). (E) Increasing concentrations of p210 BCR-ABL were cotransfected with 0.1 μg of FA-Csk plasmid (kinase-active or inactive CskGFP-FAT, indicated by closed or open circles, respectively) per well. In all experiments shown in panels A to E, cells were cultured for 24 h after transfection in DMEM containing 10% calf serum before fixation. The values in panels B to E (means ± standard deviations in a triplicate experiment) indicate the numbers of transfected (GFP-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective. All experiments were repeated at least three times.

FIG. 4.

CrkL/Crk and their SH3 binding protein, C3G, mediate an Src family-dependent adhesion mechanism. (A) Experiments similar to those illustrated in Fig. 3B were carried out with human CRKL or mouse Crk (cotransfection with kinase-active or -inactive CskGFP-FAT, indicated by closed or open circles, respectively). (B) Experiments similar to those described above were carried out with C3G-F or DOCK180-F (farnesylated mutant of C3G or DOCK180, respectively; Myc-tagged). The Western blot shows expression levels of DOCK180 in NIH 3T3 cells transfected with 0, 0.1, or 1 μg of plasmid in a 60-mm-diameter plate (each dose corresponds, from left to right, to 0, 0.01, and 0.1 μg of plasmid in 1 well of a 24-well plate as performed in detachment assays, respectively). In all experiments, cells were cultured for 24 h after transfection in DMEM containing 10% calf serum before fixation. The values (means ± standard deviations from a triplicate experiment) indicate the number of transfected (GFP-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective in panels A and B. All experiments were repeated at least three times.

FIG. 5.

Involvement of Rap1, Ras, and Raf1 in Src family-dependent adhesion. Experiments similar to those illustrated in Fig. 3B were carried out. The values (means ± standard deviations from triplicate experiments) indicate the number of transfected (GFP-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective. (A) Activated Rap1 (Rap1b 12V) was cotransfected with kinase-active or -inactive CskGFP-FAT (closed or open circles, respectively) by lipofection into NIH 3T3 cells plated on fibronectin. (B) SrcY529F (0.05 μg per well) and/or a dominant-negative Rap1 (0.1 μg of Rap1b 17N) were cotransfected with FA-Csk (0.1 μg of CskGFP-FAT; kinase active or inactive) by lipofection into NIH 3T3 cells plated on fibronectin. Symbols + or − below the panel indicate the presence or absence of the transgene, respectively. (C) Increasing concentrations of a dominant negative Ras (Ras 17N) or dominant negative Raf1 (Raf-C4; the N-terminal half without the kinase domain of Raf1) were cotransfected with FA-Csk (0.1 μg of kinase-active or -inactive CskGFP-FAT, indicated by closed or open circles, respectively) into NIH 3T3 cells plated on fibronectin by lipofection. (D) A fixed amount (0.2 μg) of Ras 61L (closed circles), Raf-BXB (triangles), or empty vector control (MIGR1; open circles) was cotransfected with increasing concentrations of SrcY529F into NIH 3T3 cells plated on fibronectin-coated coverslips by lipofection. All experiments were repeated at least three times.

FIG.6.

Src kinases are essential for sustained integrin activation and FA structures. (A) Effects of the β₁ integrin affinity-activating antibody TS2/16 on cell spreading in WI-38 cells expressing kinase-active or -inactive CskGFP-FAT [FA-Csk(+) or FA-Csk(−), respectively]. To prevent cell detachment while expressing FA-Csk, cells were initially transfected on PLL before TS2/16 treatment and replating. The values (means ± standard deviations) indicate the numbers of transfected (GFP-positive) cells attached (with or without spread morphology) or cells spread in 30 random fields under a 25× objective lens 1 h after replating on to fibronectin. Asterisks indicate groups significantly different from the corresponding groups without TS2/16 treatment (t test; *, P < 0.001; **, P < 0.005). (B) The subcellular localization of kinase-active or -inactive CskGFP-FAT [FA-Csk(+) or (−), respectively] was determined 2 h after replating with or without TS2/16 pretreatment. (C) The subcellular localization of GFP-LIM was determined in WI38 cells expressing activated Raf1 (Raf-BXB) 2 h after replating with or without TS2/16 pretreatment. WI38 cells were cotransfected with GFP-LIM and Raf-BXB as described in Materials and Methods. (D) The subcellular localization of kinase-active or -inactive CskGFP-FAT was determined in Rap1 12V-rescued NIH 3T3 cells 18 h after plating onto fibronectin. Cells were coelectroporated with 2 μg of Rap1 12V vector and 20 μg of CskGFP-FAT vector. Colocalization of paxillin with CskGFP-FAT was confirmed by immunofluorescent staining (not shown).

FIG. 7.

Time-lapse observation of FA structures in living cells expressing kinase-active CskGFP-FAT on fibronectin. MEFs that express GFP fluorescence were observed for an extended time. DIC and GFP fluorescence images were recorded. Asterisks in the DIC images indicate an example of a GFP-positive cell. A part of the merged image (white rectangle) is enlarged to show details (enlarged). White arrowheads indicate an example of typical changes that occur in FA structures. Note that the distal part of the FA complex (red) was gone and the proximal portion of the FA complex was extended (green) between 10 and 11 h. Yellow indicates overlap in the images taken at 10 and 11 h.

FIG. 8.

The effect of FA-Csk on cell adhesion is independent of caspase-mediated apoptosis. (A) The effect of a membrane-anchored caspase domain of caspase 8 (CD8-C) on cell adhesion was determined with or without a caspase 3 inhibitor. After lipofection of NIH 3T3 cells (plated on fibronectin) with various amounts of pCEFL-CD8-C and 0.1 μg of a GFP expression plasmid, cells were cultured for 18 h with or without 8 μg of Z-DEVD-FMK (Calbiochem) per ml in DMEM containing 10% calf serum. (B) The effect of the caspase inhibitor Z-DEVD-FMK on cell detachment caused by FA-Csk(+) was determined. After lipofection of NIH 3T3 cells (plated on fibronectin) with kinase-active or inactive CskGFP-FAT, cells were cultured for 18 h in DMEM containing 10% calf serum and various doses of the caspase inhibitor. (C) Effect of activated (myristoylated) Akt on cell detachment caused by CD8-C expression. The number of GFP-positive cells remaining on coverslips in the group transfected with 0.3 μg of pCEFL-CD8-C is shown as a percentage of control (CTR) (that is, the mock-transfected group represents 100%) in parental NIH 3T3 cells as well as in those overexpressing a myristoylated mutant of Akt (NIH 3T3-myrAKT). All groups were cotransfected with a GFP expression vector. The Western blot above the bars shows the level of total Akt (endogenous and transfected) in NIH 3T3-myrAKT cells (right lane) compared to that of endogenous Akt in parental NIH 3T3 cells (left lane). (D) Effect of FA-Csk (0.1 μg of kinase-active or -inactive CskGFP-FAT plasmid) in NIH 3T3-myrAKT cells plated on fibronectin. (A to D) Values (means ± standard deviations from a triplicate experiment) indicate the numbers of transfected (GFP fluorescence-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective (for panel C, values are given as percentages of control cells as noted above). (E) DNA fragmentation and chromatin condensation were determined for NIH 3T3 cells expressing FA-Csk(+) or FA-Csk(−) (kinase-active or -inactive CskGFP-FAT) plated on PLL/fibronectin double-coated coverslips 18 h after transfection. Each column shows one field of cells in three different channels (TUNEL, DAPI, and GFP signals). The arrows indicate the GFP-positive transfected cell in each group. Even after blocking endogenous biotinylated proteins, some background staining can be seen in mitochondria in TUNEL staining. The nuclei were stained with DAPI. All experiments were repeated at least three times (A to E).