Identification of Src-Specific Phosphorylation Site on Focal Adhesion Kinase: Dissection of the Role of Src SH2 and Catalytic Functions and Their Consequences for Tumor Cell Behavior

Abstract

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr 407 , Tyr 576 , Tyr 577 , and Tyr 861 , which are considered to be Src kinase substrates. This Src kinase-independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes-deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr 925 of FAK was not evident in SrcMF-or Src251-expressing cells, and lack of Src kinasedependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors. (Cancer Res 2005; 65(4): 1335-42)

Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells

“…We and others have previously demonstrated that Src kinase promotes cellular invasion through a mechanism involving activation of FAK [11,19,59]. Importantly, dual treatment of both MCF7 and T47D cells, in addition to suppressing Src activity, also consistently suppressed the activity of the Src-associated molecule FAK a central component of the cells' migratory machinery.…”

Copine-III interacts with ErbB2 and promotes tumor cell migration

“…Moreover, in Copine-III KD cells we observe decreased Src activity and decreased FAK phosphorylation at the Src site (pY925). Phosphorylation of Tyr925FAK by Src is required for proper FA turnover and cell migration (Brunton et al, 2005). A RACK1-Src association has been described (Miller et al, 2004).…”

“…In contrast, there was little or no increase in pTyr418Src levels in the two Copine-III KD pools (Figure 7f). Moreover, the level of pTyr925FAK, a site that has been shown to require Src kinase activity to become phosphorylated (Brunton et al, 2005), also increased in HRG-treated control cultures, whereas little or no increase was observed in Copine-III KD pools (Figure 7f), attesting to the decrease in Src activity. In contrast to the effects on Src kinase activity, activation of the Akt and Erk pathways was similar in response to HRG treatment in the Copine-III KD cultures and the control cells (Figure 7f).…”

Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases

“…Signaling through Src has been proposed to be involved in regulating cell migration and invasion by integrins (Sieg et al, 2000;Hauck et al, 2002;Hsia et al, 2003;Frame, 2004;Westhoff et al, 2004;Brunton et al, 2005). Two downstream targets of Src, FAK and p130Cas, have been proposed to be required for integrin-mediated migration (Vuori et al, 1996;Klemke et al, 1998;Hauck et al, 2002).…”

“…Furthermore, reexpression of CD82 did not alter signaling to Erk or to Akt ( Figure S3B). Both integrins and c-Met have been shown to be effective in activating Src (Rahimi et al, Hauck et al, 2002;Hsia et al, 2003;Frame, 2004;Westhoff et al, 2004;Brunton et al, 2005). To determine if CD82 is involved in signaling to Src, we investigated Src activation following integrin engagement in CD82-expressing cells.…”