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. 1998 May;18(5):3044-58.
doi: 10.1128/mcb.18.5.3044.

Activation of Rho-dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

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Free PMC article

Activation of Rho-dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

Z F Altun-Gultekin et al. Mol Cell Biol. .
Free PMC article

Abstract

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

Figures

FIG. 1
FIG. 1
Quantitative changes in the surface area of v-Crk-expressing cells in the presence and absence of serum and upon treatment with LPA or NGF. (B) When maintained in 15% serum-containing medium, the majority of v-Crk-expressing cells became flattened. (C) These cells were separated from the minor subset of cells that remained round in serum-containing medium, as described in the text. (A) When serum was withdrawn, the flattened v-Crk cells reverted to the round phenotype. (D) Bar graphs show the changes in mean surface areas of cells under various treatment conditions. The standard errors are also shown. The surface areas were measured by scanning photographs of random fields of cells and measuring the scanned images of individual cells on each photograph as described in Materials and Methods. (Bar 1) v-Crk cells that remained round in 15% serum; surface area, 107 + 73 μm2 (n = 51). (Bar 2) Flat v-Crk cells in medium without serum; mean surface area, 137 + 7 μm2 (n = 40). (Bar 3) Flat v-Crk cells that were maintained in the absence of serum and later treated with 1 μm LPA; mean surface area, 411 + 50 μm2 (n = 46). (Bar 4) Flat v-Crk cells in 15% serum-containing medium; mean surface area, 617 + 42 μm2 (n = 45). (Bar 5) Flat v-Crk cells maintained in 15% serum and then treated with 100 ng of NGF per ml for 5 days; mean surface area, 1,276 + 175 μm2 (n = 52). The differences between mean surface areas of round versus flat v-Crk cells in 15% serum (t test, P < 0.001), flat v-Crk cells in the absence versus the presence of 15% serum (t test, P < 0.001), flat v-Crk cells in the absence of serum versus after LPA treatment (t test, P < 0.001), and v-Crk cells kept in serum versus after additional NGF treatment (t test, P < 0.001) were significant.
FIG. 2
FIG. 2
v-Crk causes serum-induced cell flattening and lamellipodium formation in PC12 cells through the activation of Rho. v-Crk-expressing PC12 cells were grown in the presence (A and C) or absence (B and D) of 15% serum-containing medium. (A) Representative shape changes of PC12 cells expressing v-Crk when cultured in 15% serum. Cells flatten out and grow lamellipodia along their edges (arrows). (B) When cells are serum starved for 12 h, no flat cells are observed. (C) Cells maintained in 15% serum were treated with 100 μg of C3 exoenzyme per ml for 12 h. (D) Cells were maintained in serum-free medium for 12 h and then treated with 1 μM LPA for 3 h. Magnification, ×26.5. Bar, 50 μm.
FIG. 3
FIG. 3
Serum- and LPA-induced cell flattening is blocked by mutation in the v-Crk SH2 or SH3 domain. (A) R273N–v-Crk (SH2 mutant) is defective in binding tyrosine-phosphorylated paxillin. D386DRHAD–v-Crk, R273N–v-Crk, or wild-type (WT) v-Crk-expressing cells were kept in the presence or absence of NGF, and the resulting detergent lysates were immunoprecipitated with anti-Gag antibodies and subjected to Western blotting with antipaxillin MAb (arrow). (B) Linker insertion mutations in the Crk SH3 domain disrupt binding to proline-rich peptides derived from the Crk-binding region of C3G. GST or GST fusion proteins containing either wild-type v-Crk SH3 or D386DRHAD SH3 domains were labeled with [32P]orthophosphate (see Materials and Methods). To quantify the binding of the 32P-labeled GST proteins to C3G-derived peptides, 3.5 μg of GST (lanes 1), GST-C3GCB1 (SPPPALPPKKRG) (lanes 2), or GST-C3GK10L (SPPPALPPKLRG) (lanes 3) was electrophoretically resolved in a 13% acrylamide gel and transferred to Immobilon P, and membrane strips were incubated with [32P]GST, [32P]GST–v-Crk SH3, or [32P]GST–D386DRHAD–v-Crk SH3 overnight at 4°C. After being washed, the filters were exposed to X-ray film (autoradiogram) or excised and counted in a β-counter (histograms). (C) Morphological responses of cells expressing v-Crk mutants towards LPA. Native PC12 cells (panels i and iv), R273N–v-Crk cells (panels ii and v), or D386DRHAD–v-Crk cells (panels iii and vi) were grown in serum-free medium for 12 h (panels i to iii) and then treated with 1 μM LPA for 3 h (panels iv to vi). Similar results were obtained with serum (not shown). BSP v-CrkSH3 is th esame as D386DRHAD–v-CrkSH3. The structures and positions of SH2 and SH3 mutations are indicated. Magnification, ×32.
FIG. 4
FIG. 4
LPA and serum antagonize the neurite-promoting effects of v-Crk unless serum is withdrawn. PC12 cells (A), D386DRHAD–v-Crk cells (B), R273N–v-Crk cells (C), or v-Crk cells (E and F) were cultured for 5 days in 15% serum containing NGF (100 ng/ml). v-Crk PC12 cells exhibiting broadened lamellipodia and flattening in panels E and F are indicated by solid arrows. In panel D, serum was removed from v-Crk-expressing cells for 12 h and the cells were cultured in serum-free medium containing 100 ng of NGF per ml. Note the absence of somal flattening in these cells (compare panel D with panels E and F).
FIG. 5
FIG. 5
PI(4,5)P2 production in PC12 cells expressing v-Crk and v-Crk mutants. Native PC12 cells, v-Crk cells (clones V15F and V1F), R273N–v-Crk cells, or D386DRHAD–v-Crk cells were maintained in 15% serum, after which they were briefly starved and incubated overnight with 20 μCi of [32P]orthophosphate. Extracts were prepared from adherent cells and normalized for cellular protein, and the resulting radiolabeled lipids were subjected to TLC. 32P-labeled PIP2 was deacylated and quantified with a PhosphorImager. Values were normalized to the value in native PC12 cells (designated 100%) and are expressed as the mean and standard error (P < 0.05 between V15F and V1F and native cells, indicated by asterisk) of four independent experiments. HPLC analysis on extracted lipids demonstrated that only PI(4,5)P2, and not PI(3,4)P2, was produced in these cells.
FIG. 6
FIG. 6
v-Crk activates p160ROCK by transient transfection into 293T cells. (A) 293T cells were transiently cotransfected by the calcium phosphate method with 2 μg of pCMX-myc-tagged ROCK (lanes 2 to 4) and either 2 μg of pMEXneo (control, lane 2), pMEXneo–v-crk (lane 3), or activated pcEXV-V14rhoA (lane 4) for 3 h. After an additional 36 h in 15% serum, the cells were washed and immunoprecipitated with anti-Myc MAb. In the top panel, the level of p160ROCK activity was determined in an in vitro kinase assay with 5 μg of histone H1 as a substrate. The expression levels of myc-p160ROCK and v-Crk, determined by Western blotting, are shown in the respective bottom panels. (B) A replicate experiment, except that cells were also transfected with 2 μg of pMEXneo–R273N–v-crk plasmid DNA. (C) Effects of dominant negative Rho or Rac on the v-Crk-induced activation of MycROCK. Transfections were performed as above, except that 2 μg of pEXV-N19rhoA (lane 2) or pEXV-N17rac1 (lane 3) was used as the control for Rho specificity.
FIG. 7
FIG. 7
The Rac/Cdc42-activated protein kinases PAK and S6 kinase are not hyperactivated by v-Crk. (A and B) Native, R273N–v-Crk-expressing, D386DHRAD–v-Crk-expressing, or v-Crk-expressing PC12 cells were serum starved for 12 h (lanes 1) and then stimulated with 15% serum (lanes 2) or 15% serum plus 50 ng of NGF per ml (lanes 3) for an additional 30 min. The cells were washed, detergent lysates were prepared, and 500 μg of total cellular protein was immunoprecipitated with either anti-PAK65 polyclonal antibody (A) or anti-S6 kinase polyclonal antibody (B). The kinase activity was determined after incorporation of 32P into myelin basic protein (MBP) or S6 proteins, respectively (indicated by arrows). (C) 293T cells were transiently transfected as described in the legend to Fig. 6A, except that 2 μg of pJ3H-PAK DNA and 2 μg of either pEXV-V12rac1 (lane 2), pMex-v-Crk (lane 3), or pMEXneo control (lane 4) were used. The cells were lysed and immunoprecipitated with anti-HA antibodies, and kinase activity was measured with 5 μg of myelin basic protein as the substrate (arrow). The expression of HA-PAK by using anti-HA antibody is indicated in the bottom panel (arrow). (D) Activation of cellular JNK by v-Crk and mutant v-Crk. PC12 cells and wild-type or mutant v-Crk PC12 cells were treated as above and immunoprecipitated with anti-JNK antibodies. Kinase activity was measured after incorporation of 32P into the GST–c-Jun 1-79 substrate (arrow).
FIG. 8
FIG. 8
v-Crk promotes the formation of stress fibers and focal adhesions that contain vinculin and paxillin. Native PC12 cells (A and B) or V15F cells (C to H) were cultured in 15% serum on poly(l-lysine)-coated glass coverslips. The cells were immunostained with antipaxillin (A and C) or antivinculin (E) antibodies and counterstained with rhodamine-conjugated phalloidin (B, D, and F) to detect actin microfilaments. Solid arrows demarcate areas of well-defined peripheral focal adhesions that end in stress fibers (compare the arrows in panels C and D and those in panels E and F). Panels G and H show merged composites of vinculin and actin (G) and paxillin and actin (H). Focal adhesions that stained yellow (arrows) indicate regions of colocalization. Magnification, ×91. Scale bar, 3.3 μm in panels A and B; 10 μm in panels C through H.
FIG. 9
FIG. 9
v-Crk elevates the expression and tyrosine phosphorylation of FAK. (A) PC12 or V15F cells were maintained in low serum (3%) for 4 h and then stimulated with either 15% serum or 15% serum plus 100 ng of NGF per ml for 5 or 30 min. Lysates were normalized for cellular protein, immunoprecipitated with anti-FAK MAb, and subjected to Western blotting with anti-PY (top panel). In the bottom panel, the same blot was stripped and reprobed with anti-FAK antibody. The constitutive level of tyrosine phosphorylation in the presence of low serum probably reflects the fact that the cells were kept in low serum for only 4 h, at which time they were still predominantly flattened. (B) Tyrosine phosphorylation of FAK in V15F cells is dependent on cell adhesion and Rho activation (lanes 2 to 7). The cells were treated as in panel A, except in lane 4, where V15F cells were treated with EDTA and grown in suspension in spinner flasks with 15% serum for 4 h. In lanes 5 to 7, V15F cells were treated in the absence (lane 5) or presence (lanes 6 and 7) of C3 toxin for 12 or 24 h. The levels and tyrosine phosphorylation of FAK are indicated.
FIG. 10
FIG. 10
The expression levels of FAK, paxillin, α3-integrin, and a higher-molecular-weight form of β1-integrin are selectively increased in v-Crk-expressing cells. D386DRHAD–v-Crk (lane 1), native PC12 cells (lane 2), R273N–v-Crk (lanes 3), and round or flat forms of two independent lines of v-Crk cells, V1 flat cells (lanes 4), V1 round cells (lanes 5), V15 flat cells (lanes 6), and V15 round cells (lanes 7), were maintained in 15% serum. Round cells were obtained by slight manual trituration of the cells, which lifted off the plate easily, leaving the flattened cells which contained focal adhesions still attached. After cell lysis, protein concentrations were normalized and 100 μg of total cellular protein was analyzed by SDS-PAGE and Western blotting with specific antibodies to Gag (v-Crk), vinculin, talin, p130cas, β1-integrin, paxillin, FAK, α3-integrin, and αv-integrin, as indicated. Expression of FAK was increased in both forms of v-Crk cells, although to a lesser degree in the round cells (compare lanes 4 and 6, and compare lanes 5 and 7). α1-Integrin was detected after cell surface biotinylation followed by immunoprecipitation and immunoblotting with horseradish peroxidase-conjugated streptavidin. Although another strain of PC12 cells expressed α1-integrin at high levels (lane 8), the PC12 cells used in this study as well as v-Crk and mutant v-Crk-expressing cells showed very low detectable levels of α1-integrin.
FIG. 11
FIG. 11
v-Crk confers resistance to apoptosis during serum withdrawal. Native PC12 or V15F PC12 cells were cultured in 15% serum and transferred to serum-free medium for up to 36 h. Live versus dead cells were scored by a two-color fluorescence assay involving ethidium homodimer and calcein AM. Data show the mean and standard error for three independent experiments.

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