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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

FAK expression and phosphorylation during development.(A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1st column), early gastrula (2nd column) and late gastrula embryos (3rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 µm.
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pone-0042577-g001: FAK expression and phosphorylation during development.(A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1st column), early gastrula (2nd column) and late gastrula embryos (3rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 µm.

Mentions: Xenopus FAK was originally cloned by Zhang et al. and its expression was analyzed in detail by the DeSimone group. It was determined that FAK is expressed maternally and that elevated levels are found in the highly mophogenetically active mesodermal tissues and in addition at the somitic boundaries. Additionally, increased levels of expression and phosphorylation of FAK were observed during gastrulation indicating that FAK may be involved in regulating embryonic cell adhesive behaviour and morphogenesis [31], [32]. In an effort to better characterize the spatiotemporal expression and phosphorylation of FAK in the embryo we examined the endogenous levels of phosphorylation on tyrosines 397, 576 and 861 in western blotting (Figure 1A) and whole mount immunofluorescence experiments (Figure 1B) using previously characterized antibodies against the phosphorylated forms of the above sites. As shown in Figure 1 all three sites are phosphorylated in all developmental stages we examined including pre-gastrula stages. Phosphorylation of tyrosine 397 follows a similar pattern to what was reported by Hens and DeSimone for total phospho-FAK, ie phosphorylation increases during development with an increase observed during gastrulation [32]. A similar increase is observed for phosphorylation of tyrosines 576 and 861 however for these two sites a drop is observed during neurulation (Figure 1A). Whole mount indirect immunofluorescence shows that phosphorylated FAK is localized at the plasma membrane of the cells (Figure 1B) suggesting that FAK activation takes place at the plasma membrane as expected. Elevated levels of phosphorylated FAK are seen in the highly morphogenetic mesodermal tissues suggesting a possible involvement of FAK in these movements (Figure 1B). Examination of the localization pattern of tyrosine phosphorylated paxillin (Y-31) at these stages reveals a very similar pattern to that of phosphorylated FAK (Figure 1B, 4th row). High magnification optical sections at the blastopore lip reveal that the mesoderm contains much higher levels of phosphorylated FAK (Figure 2A, white arrow) compared to the adjacent endoderm of the blastopore (Figure 2A, red arrow) as well as the single layer of endodermal cells that will line the archenteron and surround the mesoderm (Figure 2A, white arrowheads). In addition, the superficial cells of the ectoderm on the animal cap display lower levels of FAK phosphorylation compared to the deep cells (Figure 2B). The detection of phosphorylated FAK prior to gastrulation including early blastula stages and the presence of phosphorylated FAK on the apical surface of superficial cells is surprising (Figure 1B, 1st column and Figure 2C). Prior to gastrulation there is no fibronectin secretion [33], and no laminin expression [34]. In addition, cells from Xenopus embryos are unable to spread or migrate on fibronectin prior to gastrulation [35]. These taken together suggest that there is little, if any cell-ECM signalling at these early stages of development and thus FAK phosphorylation is most likely integrin-independent. This notion is also supported by the presence of phosphorylated FAK at the apical surface of superficial blastomeres which are clearly not exposed to the ECM (Figure 2C). The apical surface of each superficial cell is isolated from the basolateral region with tight junctions which prevent diffusion of membrane bound molecules between the two areas [36]. Thus it is likely that activated FAK at the apical surface of these cells is exclusively activated through mechanisms independent of integrin signalling.


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)

FAK expression and phosphorylation during development.(A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1st column), early gastrula (2nd column) and late gastrula embryos (3rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042577-g001: FAK expression and phosphorylation during development.(A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1st column), early gastrula (2nd column) and late gastrula embryos (3rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 µm.
Mentions: Xenopus FAK was originally cloned by Zhang et al. and its expression was analyzed in detail by the DeSimone group. It was determined that FAK is expressed maternally and that elevated levels are found in the highly mophogenetically active mesodermal tissues and in addition at the somitic boundaries. Additionally, increased levels of expression and phosphorylation of FAK were observed during gastrulation indicating that FAK may be involved in regulating embryonic cell adhesive behaviour and morphogenesis [31], [32]. In an effort to better characterize the spatiotemporal expression and phosphorylation of FAK in the embryo we examined the endogenous levels of phosphorylation on tyrosines 397, 576 and 861 in western blotting (Figure 1A) and whole mount immunofluorescence experiments (Figure 1B) using previously characterized antibodies against the phosphorylated forms of the above sites. As shown in Figure 1 all three sites are phosphorylated in all developmental stages we examined including pre-gastrula stages. Phosphorylation of tyrosine 397 follows a similar pattern to what was reported by Hens and DeSimone for total phospho-FAK, ie phosphorylation increases during development with an increase observed during gastrulation [32]. A similar increase is observed for phosphorylation of tyrosines 576 and 861 however for these two sites a drop is observed during neurulation (Figure 1A). Whole mount indirect immunofluorescence shows that phosphorylated FAK is localized at the plasma membrane of the cells (Figure 1B) suggesting that FAK activation takes place at the plasma membrane as expected. Elevated levels of phosphorylated FAK are seen in the highly morphogenetic mesodermal tissues suggesting a possible involvement of FAK in these movements (Figure 1B). Examination of the localization pattern of tyrosine phosphorylated paxillin (Y-31) at these stages reveals a very similar pattern to that of phosphorylated FAK (Figure 1B, 4th row). High magnification optical sections at the blastopore lip reveal that the mesoderm contains much higher levels of phosphorylated FAK (Figure 2A, white arrow) compared to the adjacent endoderm of the blastopore (Figure 2A, red arrow) as well as the single layer of endodermal cells that will line the archenteron and surround the mesoderm (Figure 2A, white arrowheads). In addition, the superficial cells of the ectoderm on the animal cap display lower levels of FAK phosphorylation compared to the deep cells (Figure 2B). The detection of phosphorylated FAK prior to gastrulation including early blastula stages and the presence of phosphorylated FAK on the apical surface of superficial cells is surprising (Figure 1B, 1st column and Figure 2C). Prior to gastrulation there is no fibronectin secretion [33], and no laminin expression [34]. In addition, cells from Xenopus embryos are unable to spread or migrate on fibronectin prior to gastrulation [35]. These taken together suggest that there is little, if any cell-ECM signalling at these early stages of development and thus FAK phosphorylation is most likely integrin-independent. This notion is also supported by the presence of phosphorylated FAK at the apical surface of superficial blastomeres which are clearly not exposed to the ECM (Figure 2C). The apical surface of each superficial cell is isolated from the basolateral region with tight junctions which prevent diffusion of membrane bound molecules between the two areas [36]. Thus it is likely that activated FAK at the apical surface of these cells is exclusively activated through mechanisms independent of integrin signalling.

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