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Activation of endogenous FAK via expression of its amino terminal domain in Xenopus embryos.

Petridou NI, Stylianou P, Christodoulou N, Rhoads D, Guan JL, Skourides PA - PLoS ONE (2012)

Bottom Line: It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place.Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.

ABSTRACT

Background: The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.

Principal findings: Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK's localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.

Conclusions: Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

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Related in: MedlinePlus

The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner.HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A–D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E–H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I–L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M–P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R–U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 50 µm, (R) 20 µm.
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pone-0042577-g005: The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner.HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A–D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E–H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I–L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M–P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R–U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 50 µm, (R) 20 µm.

Mentions: To further explore the role of the FERM domain in the activation of FAK in Xenopus we examined the effects of FERM domain overexpression in the developing embryo. Embryos were injected with the FERM domain either at the animal pole or at the two dorsal blastomeres of four cell stage embryos and were allowed to develop to tadpole stages. FERM expressing embryos developed normally and were identical to controls suggesting that FAK function was not being affected (data not shown). To examine the effects of FERM expression on endogenous FAK the experiment was repeated and embryos were either fixed or lysed at gastrula stage. As shown in Figure 5, cells expressing FERM, show elevated levels of endogenous phosphorylated FAK on tyrosines 576 and 861 compared to un-injected neighbouring cells suggesting that FERM expression leads to activation of endogenous FAK (Figure 5A–D, 5I–L respectively, red stars: injected cells, white stars: control cells). This is a surprising finding and several experiments were carried out to ensure that the phospho-FAK antibodies used were in fact specific in this context. These control experiments are described in detail in the methods section and presented in Figure S1. To confirm the activation of FAK in FERM overexpressing embryos western blotting experiments and densitometry analysis were carried out. Lysates from FERM injected embryos contain comparable levels of total FAK as controls but elevated phosphorylation on tyrosines 397, 576 and 861 (Figure 5Q). It should be noted that only a subset of the cells in the embryo are expressing the construct (∼50%) so the upregulation is effectively underestimated in western blotting experiments. In addition blotting of the HA-FERM with an anti P-Y397 antibody shows that the exogenously expressed protein is phosphorylated on tyrosine 397 in agreement with previously published work (Figure 5Q, 2nd row) [25], [42]. Expression of the N-terminus of FAK has been previously shown to block integrin-dependent FAK activation [27], [28]. To preclude the possibility that the observed effect is Xenopus specific we expressed the FERM domain in Xenopus A6 cells and examined the effects on endogenous levels of phospho-576 via indirect immunofluorescence. As shown in Figure 5 (R–U) FERM expression in Xenopus adherent cells results in a moderate reduction (compared to the more drastic effects of FRNK expression, Figure 3M–P) of phospho-576 levels indicating that FERM does in fact block FAK activation in cultured Xenopus cells. The opposite results obtained in vivo and in vitro with regard to the effects of FERM expression may be explained by a differential effect of FERM expression in integrin vs non integrin-based activation of FAK. Expression of the N-terminus of FAK in FAK cells has been shown to actually partially rescue the EGF induced cell migration defect suggesting that the FERM domain can partially transduce growth factor based signals autonomously supporting this possibility [25].


Activation of endogenous FAK via expression of its amino terminal domain in Xenopus embryos.

Petridou NI, Stylianou P, Christodoulou N, Rhoads D, Guan JL, Skourides PA - PLoS ONE (2012)

The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner.HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A–D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E–H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I–L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M–P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R–U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 50 µm, (R) 20 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412797&req=5

pone-0042577-g005: The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner.HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A–D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E–H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I–L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M–P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R–U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 50 µm, (R) 20 µm.
Mentions: To further explore the role of the FERM domain in the activation of FAK in Xenopus we examined the effects of FERM domain overexpression in the developing embryo. Embryos were injected with the FERM domain either at the animal pole or at the two dorsal blastomeres of four cell stage embryos and were allowed to develop to tadpole stages. FERM expressing embryos developed normally and were identical to controls suggesting that FAK function was not being affected (data not shown). To examine the effects of FERM expression on endogenous FAK the experiment was repeated and embryos were either fixed or lysed at gastrula stage. As shown in Figure 5, cells expressing FERM, show elevated levels of endogenous phosphorylated FAK on tyrosines 576 and 861 compared to un-injected neighbouring cells suggesting that FERM expression leads to activation of endogenous FAK (Figure 5A–D, 5I–L respectively, red stars: injected cells, white stars: control cells). This is a surprising finding and several experiments were carried out to ensure that the phospho-FAK antibodies used were in fact specific in this context. These control experiments are described in detail in the methods section and presented in Figure S1. To confirm the activation of FAK in FERM overexpressing embryos western blotting experiments and densitometry analysis were carried out. Lysates from FERM injected embryos contain comparable levels of total FAK as controls but elevated phosphorylation on tyrosines 397, 576 and 861 (Figure 5Q). It should be noted that only a subset of the cells in the embryo are expressing the construct (∼50%) so the upregulation is effectively underestimated in western blotting experiments. In addition blotting of the HA-FERM with an anti P-Y397 antibody shows that the exogenously expressed protein is phosphorylated on tyrosine 397 in agreement with previously published work (Figure 5Q, 2nd row) [25], [42]. Expression of the N-terminus of FAK has been previously shown to block integrin-dependent FAK activation [27], [28]. To preclude the possibility that the observed effect is Xenopus specific we expressed the FERM domain in Xenopus A6 cells and examined the effects on endogenous levels of phospho-576 via indirect immunofluorescence. As shown in Figure 5 (R–U) FERM expression in Xenopus adherent cells results in a moderate reduction (compared to the more drastic effects of FRNK expression, Figure 3M–P) of phospho-576 levels indicating that FERM does in fact block FAK activation in cultured Xenopus cells. The opposite results obtained in vivo and in vitro with regard to the effects of FERM expression may be explained by a differential effect of FERM expression in integrin vs non integrin-based activation of FAK. Expression of the N-terminus of FAK in FAK cells has been shown to actually partially rescue the EGF induced cell migration defect suggesting that the FERM domain can partially transduce growth factor based signals autonomously supporting this possibility [25].

Bottom Line: It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place.Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.

ABSTRACT

Background: The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.

Principal findings: Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK's localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.

Conclusions: Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

Show MeSH
Related in: MedlinePlus