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

Sachdev S, Bu Y, Gelman IH - BMC Cancer (2009)

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. sasachdev@yahoo.com

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.

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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, SrcpoY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, SrcpoY416 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.
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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, SrcpoY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, SrcpoY416 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.

Mentions: Many studies have demonstrated physical and functional interactions between FAK and Src in response to integrin- and growth factor-mediated signals. However, most of the studies linking FAK/Src complexes with cell motility, proliferation and cell survival in cancer progression involve adherent cell populations. Yet, many parameters of cancer biology in vivo, especially metastasis, require anchorage-independent proliferation. To elucidate the roles of adhesion and/or FAK on Src-induced oncogenesis, we developed FAK+/+ or FAK-/- MEF expressing relatively similar protein and activity levels of the v-Src oncogene (Fig. 1A). As we reported previously [27], the relative level of v-Src autophosphorylation (poY416) 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). Interestingly, overall v-Src-induced tyrosine phosphorylation of cellular substrates, as well as the total number of substrates, was slightly higher in v-Src cells kept in suspension, irrespective of FAK content (Fig. 1B, right panel). This was not due to relative increases in Src activation levels in the suspended cells (Fig. 1A). Although the gross level of Src-induced tyrosine phosphorylation is similar in the presence or absence of FAK, there are a small number of substrates whose relative phosphorylation level is either increased or decreased by FAK (Fig. 1B). Moreover, in adherent cells, there were more phosphorylated Src substrates in the absence of FAK (stars), whereas in the suspended cells, there were a roughly equal number of substrates favored in the presence (arrows) or absence (stars) of FAK. These data are consistent with the notion that FAK modulates the ability of activated Src to associate with and/or phosphorylate specific cellular substrates [27].


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

Sachdev S, Bu Y, Gelman IH - BMC Cancer (2009)

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, SrcpoY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, SrcpoY416 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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, SrcpoY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, SrcpoY416 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.
Mentions: Many studies have demonstrated physical and functional interactions between FAK and Src in response to integrin- and growth factor-mediated signals. However, most of the studies linking FAK/Src complexes with cell motility, proliferation and cell survival in cancer progression involve adherent cell populations. Yet, many parameters of cancer biology in vivo, especially metastasis, require anchorage-independent proliferation. To elucidate the roles of adhesion and/or FAK on Src-induced oncogenesis, we developed FAK+/+ or FAK-/- MEF expressing relatively similar protein and activity levels of the v-Src oncogene (Fig. 1A). As we reported previously [27], the relative level of v-Src autophosphorylation (poY416) 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). Interestingly, overall v-Src-induced tyrosine phosphorylation of cellular substrates, as well as the total number of substrates, was slightly higher in v-Src cells kept in suspension, irrespective of FAK content (Fig. 1B, right panel). This was not due to relative increases in Src activation levels in the suspended cells (Fig. 1A). Although the gross level of Src-induced tyrosine phosphorylation is similar in the presence or absence of FAK, there are a small number of substrates whose relative phosphorylation level is either increased or decreased by FAK (Fig. 1B). Moreover, in adherent cells, there were more phosphorylated Src substrates in the absence of FAK (stars), whereas in the suspended cells, there were a roughly equal number of substrates favored in the presence (arrows) or absence (stars) of FAK. These data are consistent with the notion that FAK modulates the ability of activated Src to associate with and/or phosphorylate specific cellular substrates [27].

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. sasachdev@yahoo.com

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.

Show MeSH
Related in: MedlinePlus