Abstract
BackgroundFocal 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.MethodTo 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.ResultsTotal 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-δ (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-γ1 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 paxillinY118F, but not by WT-paxillin, p120cateninY228F 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 paxillinY118.ConclusionThese data suggest that phosphorylation of Src substrates is a dynamic process, influenced temporally and spatially by factors such as FAK and adhesion.
Highlights
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
Cell lines and growth conditions FAK+/+ and FAK-/- mouse embryo fibroblasts (MEF) from a p53-/- lineage [31] transduced with v-Src or empty vector containing puror gene [30] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated bovine serum (BS)
As we reported previously [27], the relative level of v-Src autophosphorylation under adherent conditions was consistently 2-fold higher in the FAK-/- background, and this correlated with a slightly higher overall level of cellular phosphotyrosyl proteins compared to the FAK+/+ background (Fig. 1B, left panel)
Summary
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. Autophosphorylation of the focal adhesion kinase, FAK, at Y397 upon integrin-mediated activation produces an SH2-mediated binding site for Src-family tyrosine kinases, or alternatively, other signaling proteins such as phosphatidylinositol 3-kinase (PI3K), Shc, phospholipase-Cγ or Grb7 [1,2]. Current thinking suggests that the mutual activation of Src and FAK in response to growth factors, chemotactic agents and cell adhesion leads to activation of a number of downstream pathways in a spatially and temporally controlled manner [7]. Both FAK and Src have been implicated as playing major roles in cancer progression, especially relating to metastatic potential. The loss of FAK activity or expression suppresses metastatic progression in tumor xenograft models, underlining an important positive role for FAK in the development of malignancy [24]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call