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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 activates endogenous FAK leading to increased phosphorylation of FAK/Src targets.(A–D) Embryos injected with HA-FERM mRNA in two blastomeres, at the animal pole, at the four cell stage were processed for immunofluorescence using anti-HA (green) and anti-P-Y31 paxillin (red) antibodies. C is the merged image and D is an intensity color coded image. FERM expressing cells display elevated levels of phosphorylated paxillin (red stars) when compared with un-injected neighbouring cells (white stars). (E–H) Same as (A–D) but comparing phosphorylation levels of p130Cas on tyrosine 762 between FERM expressing and control cells. FERM expressing cells show elevated levels of phosphorylated p130Cas (red stars), when compared with un-injected cells (white stars). (I–L) Same as (A–D) but comparing levels of phosphorylated Akt on serine 473 between FERM expressing and control cells. Levels of phosphorylated Akt are comparable in FERM expressing cells to those of control neighboring cells. Scale bars: 20 µm.
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pone-0042577-g006: The FERM domain activates endogenous FAK leading to increased phosphorylation of FAK/Src targets.(A–D) Embryos injected with HA-FERM mRNA in two blastomeres, at the animal pole, at the four cell stage were processed for immunofluorescence using anti-HA (green) and anti-P-Y31 paxillin (red) antibodies. C is the merged image and D is an intensity color coded image. FERM expressing cells display elevated levels of phosphorylated paxillin (red stars) when compared with un-injected neighbouring cells (white stars). (E–H) Same as (A–D) but comparing phosphorylation levels of p130Cas on tyrosine 762 between FERM expressing and control cells. FERM expressing cells show elevated levels of phosphorylated p130Cas (red stars), when compared with un-injected cells (white stars). (I–L) Same as (A–D) but comparing levels of phosphorylated Akt on serine 473 between FERM expressing and control cells. Levels of phosphorylated Akt are comparable in FERM expressing cells to those of control neighboring cells. Scale bars: 20 µm.

Mentions: Since tyrosine 397 is a known Src binding site [21] and tyrosines 576 and 861 are targets of Src [18], [43] we went on to examine the possibility that an intact tyrosine 397 was required for the FERM induced activation of FAK. We generated a FERM Y397F construct and the construct was overexpressed in Xenopus embryos. As shown in Figure 5, cells expressing FERM Y397F do not display elevated levels of phosphorylation on tyrosines 576 and 861 (Figure 5E–H and M–P) suggesting that the observed activation of endogenous FAK is dependent on Src. This is also supported by the fact that in embryos treated with the Src inhibitor PP2, tyrosine phosphorylation on both 576 and 861 is severely reduced indicating that, in the developing embryo, FAK phosphorylation of the above residues is dependent on Src (Figure S1). Finally, to confirm the FERM induced activation of endogenous FAK we examined the phosphorylation status of p130Cas and paxillin, two FAK-Src downstream targets [44], [45]. Expression of FERM, but not FERM Y397F, leads to elevated phosphorylation of both paxillin and p130Cas but not of Akt which is a substrate of PI3K (Figure 6A–D, E–H, I–L) [46]. These results show that FERM expression leads to activation of endogenous FAK and subsequent phosphorylation of FAK-Src targets in a tyrosine 397 dependent manner.


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 activates endogenous FAK leading to increased phosphorylation of FAK/Src targets.(A–D) Embryos injected with HA-FERM mRNA in two blastomeres, at the animal pole, at the four cell stage were processed for immunofluorescence using anti-HA (green) and anti-P-Y31 paxillin (red) antibodies. C is the merged image and D is an intensity color coded image. FERM expressing cells display elevated levels of phosphorylated paxillin (red stars) when compared with un-injected neighbouring cells (white stars). (E–H) Same as (A–D) but comparing phosphorylation levels of p130Cas on tyrosine 762 between FERM expressing and control cells. FERM expressing cells show elevated levels of phosphorylated p130Cas (red stars), when compared with un-injected cells (white stars). (I–L) Same as (A–D) but comparing levels of phosphorylated Akt on serine 473 between FERM expressing and control cells. Levels of phosphorylated Akt are comparable in FERM expressing cells to those of control neighboring cells. Scale bars: 20 µm.
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Related In: Results  -  Collection

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

pone-0042577-g006: The FERM domain activates endogenous FAK leading to increased phosphorylation of FAK/Src targets.(A–D) Embryos injected with HA-FERM mRNA in two blastomeres, at the animal pole, at the four cell stage were processed for immunofluorescence using anti-HA (green) and anti-P-Y31 paxillin (red) antibodies. C is the merged image and D is an intensity color coded image. FERM expressing cells display elevated levels of phosphorylated paxillin (red stars) when compared with un-injected neighbouring cells (white stars). (E–H) Same as (A–D) but comparing phosphorylation levels of p130Cas on tyrosine 762 between FERM expressing and control cells. FERM expressing cells show elevated levels of phosphorylated p130Cas (red stars), when compared with un-injected cells (white stars). (I–L) Same as (A–D) but comparing levels of phosphorylated Akt on serine 473 between FERM expressing and control cells. Levels of phosphorylated Akt are comparable in FERM expressing cells to those of control neighboring cells. Scale bars: 20 µm.
Mentions: Since tyrosine 397 is a known Src binding site [21] and tyrosines 576 and 861 are targets of Src [18], [43] we went on to examine the possibility that an intact tyrosine 397 was required for the FERM induced activation of FAK. We generated a FERM Y397F construct and the construct was overexpressed in Xenopus embryos. As shown in Figure 5, cells expressing FERM Y397F do not display elevated levels of phosphorylation on tyrosines 576 and 861 (Figure 5E–H and M–P) suggesting that the observed activation of endogenous FAK is dependent on Src. This is also supported by the fact that in embryos treated with the Src inhibitor PP2, tyrosine phosphorylation on both 576 and 861 is severely reduced indicating that, in the developing embryo, FAK phosphorylation of the above residues is dependent on Src (Figure S1). Finally, to confirm the FERM induced activation of endogenous FAK we examined the phosphorylation status of p130Cas and paxillin, two FAK-Src downstream targets [44], [45]. Expression of FERM, but not FERM Y397F, leads to elevated phosphorylation of both paxillin and p130Cas but not of Akt which is a substrate of PI3K (Figure 6A–D, E–H, I–L) [46]. These results show that FERM expression leads to activation of endogenous FAK and subsequent phosphorylation of FAK-Src targets in a tyrosine 397 dependent manner.

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