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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 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.
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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.

Mentions: Table 2 shows that only a minority of the Src substrates studied were unaffected by FAK and/or adhesion. In fact, only the phosphorylation signals of Eps8, PLC-γ1 (at Y783), and Shc (at Y239/240) showed no change under these variables (Fig. 2). The phosphorylation of cortactin (at Y421) and paxillin (at Y31) was unaffected by FAK only in adherent cultures, whereas the phosphorylation of Sam68 and PKC-δ (at Y311) was unaffected by FAK only in suspension cultures. Interestingly, we observed a v-Src-induced increase in cortactin protein levels, and moreover, a slower-migrating form whose abundance was increased even more in FAK-/-[v-Src] cells. The phosphorylation of several Src substrates was enhanced by FAK, irrespective of adherence state. These include vinculin (at both the Y100 and Y1065 sites) and CAS. In contrast, the phosphorylation of p190RhoGAP, paxillin (at Y118) and Crk was favored in the absence of FAK, irrespective of adherence state. Lastly, although v-Src induced annexin II protein levels, the phosphorylation of annexin II seemed to be inhibited by v-Src in the absence of FAK.


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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651180&req=5

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.
Mentions: Table 2 shows that only a minority of the Src substrates studied were unaffected by FAK and/or adhesion. In fact, only the phosphorylation signals of Eps8, PLC-γ1 (at Y783), and Shc (at Y239/240) showed no change under these variables (Fig. 2). The phosphorylation of cortactin (at Y421) and paxillin (at Y31) was unaffected by FAK only in adherent cultures, whereas the phosphorylation of Sam68 and PKC-δ (at Y311) was unaffected by FAK only in suspension cultures. Interestingly, we observed a v-Src-induced increase in cortactin protein levels, and moreover, a slower-migrating form whose abundance was increased even more in FAK-/-[v-Src] cells. The phosphorylation of several Src substrates was enhanced by FAK, irrespective of adherence state. These include vinculin (at both the Y100 and Y1065 sites) and CAS. In contrast, the phosphorylation of p190RhoGAP, paxillin (at Y118) and Crk was favored in the absence of FAK, irrespective of adherence state. Lastly, although v-Src induced annexin II protein levels, the phosphorylation of annexin II seemed to be inhibited by v-Src in the absence of FAK.

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