Article Figures & Data
Figures
- FIG. 1.
v-Src kinase activity is required for FAK proteolysis and focal adhesion disassembly during v-Src transformation. (A) CEFs expressing ts LA29 v-Src with and without herbimycin A treatment and CEFs expressing kinase-defective ts LA29KD1 v-Src were cultured at the permissive temperature for v-Src activity for 24 h. Cell morphology and distribution of focal adhesions were evaluated by phase-contrast microscopy (magnification, ×200) and confocal microscopy (magnification, ×400) following immunocytochemistry with an antipaxillin antibody. (B) Cell lysates were prepared at sequential time points following v-Src activation (by a shift to the permissive temperature of 35°C) from CEFs expressing ts LA29 v-Src (upper panels), CEFs expressing ts LA29 v-Src following pretreatment with 1 μM herbimycin A (middle panels), and CEFs expressing ts LA29KD1 v-Src (lower panels). FAK protein levels were detected by SDS-PAGE and immunoblotting with anti-FAK antibody. Equal protein loading was determined by immunoblotting with anti-α-tubulin antibody.
- FIG. 2.
Mutation of tyrosines Y407, Y576, Y577, Y861, and Y925 of FAK inhibits v-Src-induced tyrosine phosphorylation of FAK. (A) Illustration of FAK protein domains, indicating locations of autophosphorylation and Src-regulated phosphotyrosine sites. (B) CEFs coexpressing ts LA29 v-Src with either wt- or 4-9F-FAK mutant (Myc tagged) were cultured at restrictive (−) and permissive (+) temperatures for v-Src activation. Total cell lysates were prepared from these cells, followed by IP with anti-Myc antibody. Tyrosine phosphorylation of wt-FAK and 4-9F-FAK (both 125 kDa) was detected following separation of anti-Myc IPs by SDS-PAGE and immunoblotting with antiphosphotyrosine antibody (pTyr). (C) Total tyrosine phosphorylation status and specific phosphorylation of the tyrosine 397 site on wt- and 4-9F-FAK was monitored 1 h following adhesion of nontransformed cells to fibronectin. Anti-Myc IPs were separated by SDS-PAGE and immunoblotting with antiphosphotyrosine antibody and a site-specific anti-phosphotyrosine397-FAK (397Y). (D) The kinase activities of wt- and 4-9F-FAK were directly compared by kinase assay following IP with anti-Myc antibody and incubation with substrate in the presence of [γ-32P]ATP. kd-FAK was also immunoprecipitated and included in the kinase assay as a negative control. Levels of each FAK mutant used in the kinase assay were monitored by immunoblotting of immunoprecipitates with anti-FAK antibody.
- FIG. 3.
Src-induced tyrosine phosphorylation of FAK is necessary for FAK proteolysis during v-Src-induced transformation. Cell lysates were prepared at sequential time points following v-Src activation from CEFs coexpressing, Myc-tagged wt-FAK and 4-9F-FAK with ts LA29 v-Src. Protein levels of Myc-tagged wt-FAK and 4-9F-FAK were detected by SDS-PAGE and immunoblotting with anti-Myc antibody. Equal protein loading was analyzed by immunoblotting with anti-α-tubulin antibody.
- FIG. 4.
Src-induced tyrosine phosphorylation of FAK is required for focal adhesion turnover and migration of v-Src-transformed cells. (A) CEFs coexpressing Myc-tagged wt-FAK or 4-9F-FAK with ts LA29 v-Src were cultured under restrictive conditions (41°C, v-Src off) or permissive conditions (35°C, v-Src on [24 h; two representative fields are shown]) for v-Src activity. The cell morphology and distribution of Myc-tagged wt-FAK or 4-9F-FAK were evaluated by confocal microscopy (magnification, ×600) following immunocytochemistry with an anti-Myc antibody. (B) CEFs coexpressing ts LA29 v-Src with wt-FAK or 4-9F-FAK were initially cultured at the restrictive temperature of 41°C. Once cells were preconfluent, a wound was generated, and wound size was recorded at time zero. Cells were subsequently incubated for 12 h at the permissive temperature (35°C) for v-Src activation, and cell migration into the wound was analyzed by phase-contrast microscopy (magnification, ×50). The percent distance of wound closure at 12 h following wound generation was calculated from three separate areas and expressed as mean plus standard error.
- FIG. 5.
v-Src-induced tyrosine phosphorylation of FAK enhances the association of calpain 2 with FAK, p42ERK, and v-Src. (A) CEFs coexpressing ts LA29 v-Src with either wt-FAK or 4-9F-FAK were cultured at the restrictive (−) or permissive (+) temperature for v-Src activation. Total cell lysates were prepared from these cells, followed by IP with anti-calpain 2 antibody. Calpain 2-associated v-Src, phospho-p42ERK, and Myc-tagged FAK mutants were detected after SDS-PAGE, transfer to membranes and immunoblotting (top). Direct Western blots of the same total cell lysates used for IP experiments are also shown (bottom). Control IPs with normal rabbit IgG are shown on the right. (B) The fractions of wt-FAK, 4-9F-FAK, phospho-p42ERK, and v-Src coimmunoprecipitating with calpain 2 following v-Src activation in relation to total levels present in cell lysates were calculated from three separate experiments. Values were quantified by densitometry using NIH3 image software and represent mean values plus standard errors.
- FIG. 6.
4-9F-FAK impairs v-Src-induced phosphorylation of paxillin and α-actinin but not p130cas. The tyrosine phosphorylation status of paxillin, p130cas, and α-actinin was monitored prior to and 3 h following activation of v-Src in cells coexpressing wt-FAK and 4-9F-FAK. p130cas and paxillin were immunoprecipitated with specific antibodies and then separated by SDS-PAGE and immunoblotted with antiphosphotyrosine (pTyr) antibody. To detect tyrosine phosphorylation of α-actinin, antiphosphotyrosine was used to immunoprecipitate phosphorylated proteins, which were then separated by SDS-PAGE and immunoblotted with anti-α-actinin antibody.
- FIG. 7.
4-9F-FAK does not restore calpain activity in FAK−/− cells. wt-FAK and the 4-9F-FAK mutant were stably expressed in FAK−/− MEFs. (A) Total cellular levels of calpain activity were measured in cell lysates extracted from FAK−/− MEFs and FAK−/− MEFs expressing either wt-FAK or 4-9F-FAK. Calpain activity in cell extracts was determined by using a calpain activity assay kit (BioVision Inc.) that monitors fluorescence emission induced by cleavage of a specific fluorogenic peptide substrate. Calpain activity was quantified with a fluorescence plate reader, and results are expressed as relative fluorescence units (RFU). Data represent mean values plus standard errors from three separate experiments. (B) Total cell lysates were prepared from FAK−/− cells or wt-FAK- and 4-9F-FAK-reconstituted FAK−/− cells. Lysates were separated by SDS-PAGE and immunoblotted with anti-Myc, anti-calpain 2, and anti-α-tubulin antibodies.
- FIG. 8.
Reexpression of wt-FAK but not 4-9F-FAK rescues motility of FAK−/− cells and facilitates cell deadhesion. (A) A wound was generated in subconfluent monolayers of FAK−/− cells and wt-FAK- and 4-9F-FAK-reconstituted FAK−/− cells. Wound size was recorded immediately following wound generation (time zero), and cell migration into the wound was monitored following incubation at 37°C for 12 h by phase-contrast microscopy (magnification, ×25). The percent distance of wound closure at 12 h following wound generation was calculated from three separate areas and expressed as mean plus standard error. (B) The adhesive properties of FAK−/− cells reexpressing wt- or 4-9F-FAK were assessed by monitoring trypsin-induced substrate detachment. Data represent mean values plus standard errors from three independent experiments.
- FIG. 9.
4-9F-FAK disrupts the normal F-actin/G-actin ratio and fails to rescue actin stress fiber assembly, cell attachment, and spreading of FAK−/− cells. (A) Pools of G-actin and F-actin were isolated from FAK−/− cells and FAK−/− cells expressing either wt-FAK or the 4-9F-FAK mutant. (B) Attachment of FAK−/− cells expressing either empty vector, wt-FAK, or 4-9F-FAK to fibronectin was quantified 30 and 60 min following plating. Data represent mean values with BSA background subtracted plus standard errors from three separate experiments. (C) The formation of actin stress fibers and focal adhesions in FAK−/− cells expressing wt- and 4-9F-FAK was monitored 2 h following adhesion to fibronectin. Formation of actin stress fibers and organization of the actin cytoskeleton were detected by staining fixed cells with FITC-labeled phalloidin. The distribution of focal adhesions and intracellular localization of exogenously expressed Myc-tagged FAK proteins were analyzed by immunocytochemistry with antivinculin and anti-Myc antibodies, respectively. Cells were analyzed by confocal microscopy (magnification, ×600).
- FIG. 10.
Actin stress fiber assembly and cell spreading mediated by expression of wt-FAK are dependent on calpain activity. (A) Pools of F-actin and G-actin were isolated from FAK−/− cells reconstituted with wt-FAK, with or without preincubation with the calpain inhibitor ALLN (50 μM). (B) FAK−/− cells reexpressing wt-FAK were preincubated with the calpain inhibitor ALLN (50 μM) prior to adhesion to fibronectin (for 2 h). Formation of actin stress fibers, organization of the actin cytoskeleton, and formation of focal adhesions were monitored by staining with FITC-labeled phalloidin or immunostaining with antivinculin antibody. Cells were analyzed by confocal microscopy (magnification, ×600).
- FIG. 11.
Src-induced phosphorylation of FAK is required for survival of serum-deprived v-Src-transformed fibroblasts. (A) Flow cytometric analysis of propidium iodide-stained CEFs coexpressing ts v-Src with wt-FAK or 4-9F-FAK was used to determine the proportion of cells with a sub-G1 DNA content (M1, indicative of apoptosis) in response to v-Src activation under serum deprivation conditions. (B) Flow cytometric analysis was also used to determine positive staining of annexin V-FITC (M2) to the surface of apoptotic CEFs coexpressing ts v-Src with wt-FAK or 4-9F-FAK following v-Src activation under serum deprivation conditions.
- FIG. 12.
Src-induced phosphorylation of FAK promotes optimal anchorage-independent growth of v-Src-transformed cells. CEFs expressing ts v-Src with either wt-FAK or 4-9F-FAK were cultured in soft agar at the permissive temperature (35°C) for v-Src activity. (A) Phase pictures illustrate colony formation 14 days following cell seeding (two representative fields are shown). (B) Colony formation after 14 days was quantified by counting the number of colonies per high-power field (magnification, ×25). Values represent the mean number of colonies plus standard errors from three separate fields.
- FIG. 13.
Src-induced phosphorylation of FAK coordinates adhesion dynamics, cell migration, survival, and anchorage-independent growth. A model proposing Src-induced tyrosine phosphorylation of FAK as a crucial event at adhesion sites that coordinates multiple cellular processes is shown. Src-induced phosphorylation of FAK is required for rapid actin stress fiber assembly and focal adhesion formation that promote initial cell adhesion and spreading, events that are in part mediated by calpain activity. Src-induced phosphorylation of FAK also promotes focal adhesion turnover and motility of cells via activation of calpain, most likely by linking calpain to upstream regulation by the ERK/MAPK pathway. v-Src-induced phosphorylation of FAK also promotes cell survival and anchorage-independent growth of transformed cells by substituting for integrin-derived growth and survival signals that are normally induced in response to cell-ECM adhesion and associated actin remodeling. These signals are likely to involve activation of PI 3-K/Akt, STAT3, and c-Myc, which have previously been identified as downstream mediators of both Src and FAK and are implicated in cell survival and anchorage-independent growth of transformed cells.
