Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion

Abstract

Background: Focal adhesion kinase (FAK) and Src are protein tyrosine kinases that physically and functionally interact to facilitate cancer progression by regulating oncogenic processes such as cell motility, survival, proliferation, invasiveness, and angiogenesis.

Method: To understand how FAK affects oncogenesis through the phosphorylation of cellular substrates of Src, we analyzed the phosphorylation profile of a panel of Src substrates in parental and v-Src-expressing FAK+/+ and FAK-/- mouse embryo fibroblasts, under conditions of anchorage-dependent (adherent) and -independent (suspension) growth.

Results: Total Src-induced cellular tyrosine phosphorylation as well as the number of phosphotyrosyl substrates was higher in suspension versus adherent cultures. Although the total level of Src-induced cellular phosphorylation was similar in FAK+/+ and FAK-/- backgrounds, the phosphorylation of some substrates was influenced by FAK depending on adherence state. Specifically, in the absence of FAK, Src induced higher phosphorylation of p190RhoGAP, paxillin (poY118) and Crk irrespective of adhesion state, PKC-delta (poY311), connexin-43 (poY265) and Sam68 only under adherent conditions, and p56Dok-2 (poY351) and p120catenin (poY228) only under suspension conditions. In contrast, FAK enhanced the Src-induced phosphorylation of vinculin (poY100 and poY1065) and p130CAS (poY410) irrespective of adherence state, p56Dok-2 (poY351) and p120catenin (poY228) only under adherent conditions, and connexin-43 (poY265), cortactin (poY421) and paxillin (poY31) only under suspension conditions. The Src-induced phosphorylation of Eps8, PLC-gamma 1 and Shc (poY239/poY240) were not affected by either FAK or adherence status. The enhanced anchorage-independent growth of FAK-/-[v-Src] cells was selectively decreased by expression of paxillin Y118F, but not by WT-paxillin, p120catenin Y228F or ShcY239/240F, identifying for the first time a role for paxillinpoY118 in Src-induced anchorage-independent growth. Knockdown of FAK by siRNA in the human colon cancer lines HT-25 and RKO, resulted in increased paxillinpoY118 levels under suspension conditions as well as increased anchorage-independent growth, supporting the notion that FAK attenuates anchorage-independent growth by suppressing adhesion-dependent phosphorylation of paxillin Y118.

Conclusion: These data suggest that phosphorylation of Src substrates is a dynamic process, influenced temporally and spatially by factors such as FAK and adhesion.

Figure 1

FAK- and adhesion-effects on v-Src substrate choice. (A) FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells grown in adherent or suspension conditions as described in Experimental Procedures were analyzed by IB for levels of total Src, Src^poY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, Src^poY416 and GAPDH levels in lane 2 (second from left) is not reproducible; relative Src activation levels in adherent FAK-/-[v-Src] cells are typically comparable to those in adherent FAK+/+[v-Src] cells]. (B) Anti-phosphotyrosine (MAb4G10) IB from equal protein loads of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cell lysates. M, proteins markers in kDa. Decreased Src-induced tyrosine phosphorylation events in the absence of FAK are marked by arrows whereas increased tyrosine phosphorylation events in the absence of FAK are marked by asterisks. These data are typical of at least three independent experiments. A GAPDH IB is shown below as a loading control.

Figure 2

FAK and adhesion modulate v-Src-induced phosphorylation of various Src substrates. (A) Lysates from adherent cultures of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells were probed either directly by IB for specific phosphorylated form(s) of the Src substrate proteins, total substrate protein levels or GAPDH, or probed for total phosphorylated protein by immunoprecipitating with substrate-specific Ab followed by IB for phosphotyrosine using MAb4G10. (B) Same IB or IP/IB analysis as in panel A using lysates of suspension cultures. Each of these blots is typical of at least duplicate independent experiments.

Figure 3

FAK and adhesion modulate v-Src-induced phosphorylation of p120catenin. Lysates from adherent or suspension cultures of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells were probed by IB for total p120catenin, p120catenin^poY228 or GAPDH. These data are typical of at least three independent experiments. Arrows, p120catenin identified by the p120catenin^poY228-specific Ab.

Figure 4

Enhanced v-Src-induced paxillin phosphorylation is attenuated by FAK. Lysates from adherent or suspension cultures of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells were probed by IB for total paxillin, paxillin^poY31, paxillin^poY118 or GAPDH. These data are typical of at least three independent experiments.

Figure 5

Phosphorylation of paxillin^poY118 is required for enhanced AIG by FAK-/-[v-Src] cells. (A) Left panel- IB analysis of FAK+/+[v-Src] or FAK-/-[v-Src] cell clones ("cl.") stably transfected with empty vector (--) or a GFP-paxillin^Y118F-expressing vector, probed with Abs specific for GFP, paxillin^poY118 or GAPDH. Aliquots of these cells were analyzed by anchorage-independent growth (top right) or for clonogenic efficiency (bottom right) as described in Materials and Methods. Error bars, S.E. *, P < 0.01. (B) A similar analysis as in panel A except on cells stably expressing FLAG-p120^Y228F, with IBs probed for FLAG, p120catenin^poY228 or GAPDH. Note that there is no statistical difference in the p120catenin^Y228F-mediated decrease in AIG between the FAK+/+[v-Src] and FAK-/-[v-Src] cells. (C) A similar analysis as in panel A except on cells stably expressing GST-Shc^Y239/240F, with IBs probed for GST-tag, Shc^poY239/240, or GAPDH.

Figure 6

Loss of FAK in human colon cancer cell lines leads to increased AIG and paxillin^poY188 accumulation under suspension growth conditions. (A) IB analysis of HT-25 and RKO colon cancer cells grown under adherent or suspension conditions that were incubated with either FAK or GFP siRNA for 72 h, probed for FAK, paxillin (not shown) paxillin^poY118 or GAPDH. Note that paxillin protein levels did not change under these conditions. Aliquots of the cells in panel A were analyzed for anchorage-independent growth (panel B) or for clonogenic efficiency (panel C) as described in Materials and Methods. Error bars, S.E. from triplicate plates in two independent experiments. p < 0.01. (D) Normalized AIG, based on the mean of the AIG data from panel B normalized to the mean of the survival data in panel C.