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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

FRNK does not act as a dominant negative in early Xenopus embryos.(A–D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E–H) Same as A–D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I–L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M–P) Same as I–L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R–S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 20 µm, (R–S) 40 µm.
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pone-0042577-g003: FRNK does not act as a dominant negative in early Xenopus embryos.(A–D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E–H) Same as A–D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I–L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M–P) Same as I–L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R–S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 20 µm, (R–S) 40 µm.

Mentions: To further examine the integrin component of FAK activation in the early embryo we overexpressed FRNK and examined its effect on the endogenous phosphorylation of FAK on tyrosines 397 and 576. Overexpression of FRNK has been shown to block cell spreading and tyrosine phosphorylation of endogenous FAK, paxillin, and tensin [41]. Although focal adhesion localization and thus integrin colocalization of FRNK is not the sole determinant for its dominant negative activity, it is required for this dominant negative activity suggesting that FRNK exerts its effect by blocking FAK specifically at integrin based activation sites [16]. However, as shown in Figure 3 expression of FRNK in DMZ cells has no effect on the levels of phosphorylation of FAK on tyrosines 397 and 576 during gastrulation in vivo as determined by immunofluorescence and western blotting (Figure 3 A–H, Q). This is in contrast to the clear downregulation of phosphorylation on both tyrosine 397 and 576 seen when we expressed FRNK via transfection in the Xenopus A6 cell line (Figure 3I–L, M–P respectively). In agreement with the lack of dominant negative activity FRNK fails to localize at the plasma membrane in animal cap cells unlike phosphorylated endogenous FAK which is found exclusively on the plasma membrane in these cells (Figure 3R, S). These results demonstrate a strong context dependence with regards to the dominant negative activity of FRNK and are in agreement with the possibility that the bulk of phosphorylated FAK in the early embryo results through integrin independent mechanisms.


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)

FRNK does not act as a dominant negative in early Xenopus embryos.(A–D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E–H) Same as A–D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I–L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M–P) Same as I–L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R–S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 20 µm, (R–S) 40 µm.
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pone-0042577-g003: FRNK does not act as a dominant negative in early Xenopus embryos.(A–D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E–H) Same as A–D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I–L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M–P) Same as I–L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R–S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 20 µm, (R–S) 40 µm.
Mentions: To further examine the integrin component of FAK activation in the early embryo we overexpressed FRNK and examined its effect on the endogenous phosphorylation of FAK on tyrosines 397 and 576. Overexpression of FRNK has been shown to block cell spreading and tyrosine phosphorylation of endogenous FAK, paxillin, and tensin [41]. Although focal adhesion localization and thus integrin colocalization of FRNK is not the sole determinant for its dominant negative activity, it is required for this dominant negative activity suggesting that FRNK exerts its effect by blocking FAK specifically at integrin based activation sites [16]. However, as shown in Figure 3 expression of FRNK in DMZ cells has no effect on the levels of phosphorylation of FAK on tyrosines 397 and 576 during gastrulation in vivo as determined by immunofluorescence and western blotting (Figure 3 A–H, Q). This is in contrast to the clear downregulation of phosphorylation on both tyrosine 397 and 576 seen when we expressed FRNK via transfection in the Xenopus A6 cell line (Figure 3I–L, M–P respectively). In agreement with the lack of dominant negative activity FRNK fails to localize at the plasma membrane in animal cap cells unlike phosphorylated endogenous FAK which is found exclusively on the plasma membrane in these cells (Figure 3R, S). These results demonstrate a strong context dependence with regards to the dominant negative activity of FRNK and are in agreement with the possibility that the bulk of phosphorylated FAK in the early embryo results through integrin independent mechanisms.

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