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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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The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions.Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Δ375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 µm.
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pone-0042577-g004: The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions.Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Δ375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 µm.

Mentions: The above data raised the possibility that FAK is primarily activated through integrin-independent mechanisms in the early Xenopus embryo. Since the C-terminus of FAK which is both necessary and sufficient for focal adhesion localization fails to localize at the plasma membrane of animal cap cells (Figure 3S) we postulated that the N-terminus which has been shown to bind PIP2 and growth factor receptors may in fact be the major determinant for the localization of active FAK in vivo [4], [5], [6]. To explore the role of the FERM domain in the localization of endogenous FAK on the plasma membrane, a series of constructs were generated (based on chicken FAK which shares a 91% identity and 95% similarity at the amino acid level with Xenopus FAK and conservation of all tyrosine phosphorylation sites) and examined with respect to their localization in cells of the animal pole and their ability to specifically localize to the integrin-free apical surface of these cells. Each construct was expressed as an HA tagged protein through mRNA injection at the two AP-dorsal blastomeres of four-cell stage embryos, and embryos were subsequently processed for whole mount immunofluorescence using a monoclonal anti-HA antibody. As shown in Figure 4 the FERM domain, unlike FRNK, displays strong plasma membrane localization and is also found on plasma membrane associated vesicles (Figure 4A). This pattern closely matches that of phosphorylated FAK (Figure 4B) suggesting that the FERM domain rather than the FAT domain is responsible for membrane localization of active FAK in the embryo. To further investigate the role of the FERM domain in the localization of activated FAK we examined the localization of the full length FAK K38A point mutant, in which the FERM kinase domain interaction is compromised, and compared it to that of the Δ375 mutant, which lacks the FERM domain. Both constructs are constitutively active due to the loss in the case of the K38A mutant of the FERM kinase inhibitory interaction and absence of the FERM domain in the case of the Δ375. The two constructs exhibit significant differences in terms of their ability to localize to the plasma membrane with the K38A exhibiting strong membrane localization while the Δ375 is very diffuse and appears completely absent from the plasma membrane in the cells of the animal cap (Figure 4C and D respectively).


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 is necessary and sufficient for membrane localization of FAK at integrin-free regions.Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Δ375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412797&req=5

pone-0042577-g004: The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions.Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Δ375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 µm.
Mentions: The above data raised the possibility that FAK is primarily activated through integrin-independent mechanisms in the early Xenopus embryo. Since the C-terminus of FAK which is both necessary and sufficient for focal adhesion localization fails to localize at the plasma membrane of animal cap cells (Figure 3S) we postulated that the N-terminus which has been shown to bind PIP2 and growth factor receptors may in fact be the major determinant for the localization of active FAK in vivo [4], [5], [6]. To explore the role of the FERM domain in the localization of endogenous FAK on the plasma membrane, a series of constructs were generated (based on chicken FAK which shares a 91% identity and 95% similarity at the amino acid level with Xenopus FAK and conservation of all tyrosine phosphorylation sites) and examined with respect to their localization in cells of the animal pole and their ability to specifically localize to the integrin-free apical surface of these cells. Each construct was expressed as an HA tagged protein through mRNA injection at the two AP-dorsal blastomeres of four-cell stage embryos, and embryos were subsequently processed for whole mount immunofluorescence using a monoclonal anti-HA antibody. As shown in Figure 4 the FERM domain, unlike FRNK, displays strong plasma membrane localization and is also found on plasma membrane associated vesicles (Figure 4A). This pattern closely matches that of phosphorylated FAK (Figure 4B) suggesting that the FERM domain rather than the FAT domain is responsible for membrane localization of active FAK in the embryo. To further investigate the role of the FERM domain in the localization of activated FAK we examined the localization of the full length FAK K38A point mutant, in which the FERM kinase domain interaction is compromised, and compared it to that of the Δ375 mutant, which lacks the FERM domain. Both constructs are constitutively active due to the loss in the case of the K38A mutant of the FERM kinase inhibitory interaction and absence of the FERM domain in the case of the Δ375. The two constructs exhibit significant differences in terms of their ability to localize to the plasma membrane with the K38A exhibiting strong membrane localization while the Δ375 is very diffuse and appears completely absent from the plasma membrane in the cells of the animal cap (Figure 4C and D respectively).

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