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First published online 6 January 2009
doi: 10.1242/jcs.030478

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FAK engages multiple pathways to maintain survival of fibroblasts and epithelia – differential roles for paxillin and p130Cas

Nadia K. Zouq^1,*, James A. Keeble^1,*, Jennefer Lindsay¹, Anthony J. Valentijn¹, Lu Zhang¹, Deborah Mills¹, Christopher E. Turner², Charles H. Streuli¹ and Andrew P. Gilmore^1,

¹ Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK
² Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY13210, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. Anoikis and FAK signalling in MECs and MEFs. (a) MECs and MEFs were detached from ECM for various times in the presence or absence of serum growth factors (GF). Apoptosis was quantified at each time point by examining nuclei stained with Hoechst. Apoptosis occurred in MECs in the presence or absence of GFs. MEFs were significantly protected from anoikis by the presence of GFs. (b) MECs and MEFs were left adherent or detached from ECM for various times. Cell lysates were then separated by SDS-PAGE and immunoblotted for total phosphotyrosine, phospho-FAK tyrosine 397 (Y397), phospho-paxillin Y31, total FAK and total paxillin. (c) MEFs and MECs were transiently transfected with either control vector or pCDNA3DN-FAK. MECs were cultured in the presence of GFs, whereas MEFs were grown in the presence or absence of GFs. Apoptosis was quantified 24 hours post transfection. MEFs were largely protected by GFs from apoptosis induced by DN-FAK expression. Data represent the mean of three experiments. Error bars indicate standard error of the mean.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Dominant-negative p130Cas induces apoptosis in MEFs, but not in MECs. (a) Schematic representation of Cas and the deletion mutants used: FLCas, full length; CasSH3, deletion of SH3 domain; CasSD, deletion of substrate domain; CasSH3, SH3 domain only. (b) The four p130Cas constructs, immunoblotted with either anti-CasSD, or anti-CasN17 (recognises the SH3 domain). (c) Whole-cell lysates from MEFs transiently transfected with either empty vector or vector expressing CasSH3 immunoblotted with anti-CasPY410 or anti-CasSD. Note that phosphorylation of endogenous p130Cas is virtually abolished in the CasSH3-expressing cells. (d) MECs and MEFs transiently expressing FL p130Cas, p130Cas-SH3 or DN-FAK were stained with Hoechst and apoptosis quantified. Results are the mean of three independent experiments. Error bars indicate standard error. *, Significance relative to mock control; n/s, not significant (ANOVA with Bonferroni's multiple comparison test).

View larger version (30K): [in this window] [in a new window]   Fig. 3. MyrFAK protects MECs and MEFs from anoikis. (a) Schematic diagram showing myrFAK with the v-Src myristoylation sequence and versions in which the auto-phosphorylation site at Tyr397 or Lys454 were substituted for Phe or Arg respectively (Y397F and K454R). (b) MECs transiently expressing myrFAK, myrFAKY397F or myrFAKK454R. Lysates were either separated by SDS-PAGE or immunoprecipitated (IP) with anti-V5. Lysates and IPs were immunoblotted (IB) as indicated. (c) MEFs transiently expressing myrFAK, myrFAKY397F or myrFAKK454R were immunostained with antibodies against the V5 epitope or a focal adhesion marker (anti-paxillin). (d) MECs transiently expressing myrFAK or myrFAKY397F were left adherent or detached and maintained on poly-HEMA for 30 minutes. Whole-cell lysates were immunoblotted for anti-V5, anti-phosphoFAK Y397, anti-total FAK and actin. Note that phospho-FAK Y397 is reduced in detached cells transfected with empty vector or expressing myrFAKY397F but is not reduced in cells expressing myrFAK. (e) MECs and MEFs expressing myrFAK, myrFAKY397F or myrFAKK454R, or transfected with empty vector, were detached from ECM for 24 hours. Apoptosis was quantified by nuclear morphology following Hoechst staining. MECs were maintained in the presence of growth factors, whereas MEFs were without growth factors. Data represent the mean of three experiments. Error bars indicate standard error. *, Significant reduction in apoptosis relative to the mock-transfected cells (ANOVA with Bonferroni's multiple comparison test); n/s, not significant.

View larger version (29K): [in this window] [in a new window]   Fig. 4. MyrFAK recruitment to the membrane is required for its anti-apoptotic function. (a) MECs transiently expressing myrFAK or WT-FAK were separated into cytosolic (sol) and membrane (mem) fractions. Whole-cell lysates (WCL) as well as sol and mem fractions were immunoblotted for expressed FAK (anti-V5) and mtHsp-70. Note the enrichment of myrFAK in the membrane fraction, whereas WT-FAK is only detected in the cytosol. (b) MEFs expressing myrFAK, WT-FAK or myrFAKY397F, detached from ECM and immunoblotted with the indicated antibodies. (c) MECs and MEFs expressing myrFAK, myrFAKY397F or WT-FAK, or transfected with empty vector, were detached from ECM for 24 hours. Apoptosis was quantified by nuclear morphology following Hoechst staining. MECs were maintained in the presence of growth factors, whereas MEFs were without growth factors. Data represents the mean of three experiments. Error bars indicate standard error. *, Significant reduction in apoptosis relative to the mock-transfected cells (ANOVA with Bonferroni's multiple comparison test); n/s, not significant.

View larger version (35K): [in this window] [in a new window]   Fig. 5. FAK interaction with p130Cas is required to suppress anoikis in MEFs but not in MECs. (a) Diagram of myrFAK and the proline region mutants. (b) Expression and phosphorylation of myrFAKPro1, myrFAKPro2 and myrFAKPro1/2 in MECs. Whole-cell lysates (WCL) of transiently transfected MECs were immunoblotted (IB) with anti-V5 and actin, or were immunoprecipitated (IP) with anti-V5 and the IPs immunoblotted with anti-phosphoFAKY397 or anti-FAK. (c) myrFAKPro1, myrFAKPro2 and myrFAKPro1/2 expressing MEFs were immunostained with anti-V5 and a marker for focal adhesions (anti-paxillin). (d) Deletion of the two Pro domains inhibits the ability of FAK to bind p130Cas. MEFs were co-transfected with the indicated myrFAK and p130Cas constructs. WCL were prepared and either directly immunoblotted (IB) with an anti-p130Cas antibody, or immunoprecipitated with anti-V5 antibody and then immunoblotted with anti-FAK and anti-p130Cas antibodies. (e) Suppression of anoikis in MECs and MEFs by myrFAK. MECs and MEFs transiently expressing the indicated myrFAK constructs were detached from ECM and maintained on poly-HEMA for 24 hours. Apoptosis was quantified in transfected cells by nuclear morphology. Results are the mean of three independent experiments. Error bars indicate standard error. *, Significant difference from mock-transfected cells (ANOVA with Bonferroni's multiple comparison test); n/s, no significant suppression of anoikis compared with mock-transfected cells.

View larger version (26K): [in this window] [in a new window]   Fig. 6. FAK interactions via both Y925 and paxillin are required to suppress anoikis. (a) Schematic diagram of myrFAK, myrFAKY925F and myrFAKI936/998E. (b) Expression and phosphorylation of myrFAK mutants. WCL from MECs either transfected with vector alone or transiently expressing myrFAK, myrFAKY397F, myrFAKY925F or myrFAKI936/998E were immunoprecipitated with anti-V5. Immunoprecipitates (IP) were immunoblotted (IB) with anti-FAK, and either anti-phosphoFAKY397 or anti-phosphoFAKY925, as indicated. (c) MECs transiently expressing myrFAK, myrFAKY925F or myrFAKI936/998E. Left panel: whole-cell lysates (WCL) incubated with glutathione agarose beads coated with GST-paxillin, separated by SDS-PAGE and immunoblotted with anti-V5. Right panel: cells were incubated with (+) or without (–) DSS before lysis and immunprecipitation (IP) with anti-V5, and immunoblotting (IB) with anti-paxillin or anti-V5. (d) MEFs transiently expressing myrFAKY925F or myrFAKI936/998E were immunostained with anti-V5 and anti-paxillin. myrFAKY925F was localised to focal adhesions, whereas myrFAKI936/998E was not. (e) MECs and MEFs transiently expressing myrFAK, myrFAKY925F or myrFAKI936/998E were detached from ECM and maintained on poly-HEMA for 24 hours. Apoptosis was quantified in transfected cells by nuclear morphology. Results are the mean of three independent experiments. Error bars indicate standard error. *, Significant difference from mock-transfected cells (ANOVA with Bonferroni's test); n/s indicates no significant suppression of anoikis compared with mock-transfected cells.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Dominant-negative paxillin induces apoptosis in adherent MECs. (a) Schematic diagram of GFP-paxillin constructs used. (b) Whole-cell lysates (WCL) of MECs transiently expressing GFP, GFP-paxillin or GFP-paxillinY31/118F immunoblotted with anti-GFP and anti-paxillin. (c) Wild-type and DN-paxillin localise to focal adhesions. Cells transiently expressing GFP, GFP-paxillin and GFP-paxillinY31/118F were stained with rhodamine-phalloidin and anti-phospho-paxillinY31. Note GFP-paxillin and GFP-paxillinY31/118F both localise to focal adhesions. However, the focal adhesions of GFP-paxillinY31/118F-expressing cells do not immunostain for phospho-paxillinY31. (d) Apoptosis quantified by nuclear morphology in MECs transiently expressing GFP, GFP-paxillin or GFP-paxillinY31/118F. Results are the mean of three independent experiments. Error bars indicate standard error. *, Significant increase in apoptosis compared with vector alone (ANOVA with Bonferroni's multiple comparison test); n/s indicates not significant.

View larger version (25K): [in this window] [in a new window]   Fig. 8. MyrFAK signals through multiple, independent complexes to suppress apoptosis. (a) Mock-transfected HEK293T or HEK293T cells transiently expressing myrFAK constructs were detached for 15 minutes and treated with and without the membrane permeable crosslinker DSS. Cell lysates were immunoprecipitated with anti-V5. Immunoprecipitates were immunoblotted with anti-FAK Ab and the same immunoblot re-probed with anti-paxillin Ab. *, Position of the 200 and 300 kDa complexes seen following crosslinking. (b) Suppression of anoikis in MECs expressing myrFAK, myrFAKY925F or myrFAKI936/998E alone, or both myrFAKY925F and myrFAKI936/998E. Results are the mean of three independent experiments. Error bars indicate standard error. *, Significant suppression of apoptosis compared with either myrFAKY925F or myrFAKI936/998E alone. (c) HEK293T cells transiently expressing myrFAK constructs were detached for 15 minutes as indicated. Whole-cell lysates were immunoblotted as indicated. (d) MECs and MEFs were left adherent or detached for the indicated times. MECs were left in complete growth medium. MEFs were deprived of serum growth factors, except for the indicated adherent cells. Whole-cell lysates (WCL) were prepared and immunoblotted for total Akt or phospho-Akt serine 473. (e) MECs or MEFs expressing myrFAK (+), or non-expressing controls (–), were left adherent or detached as indicated. WCL were prepared and immunoblotted for phospho-Akt serine 473, total Akt, or myrFAK (anti-V5).

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