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Cortactin tyrosine phosphorylation promotes its deacetylation and inhibits cell spreading.

Meiler E, Nieto-Pelegrín E, Martinez-Quiles N - PLoS ONE (2012)

Bottom Line: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading.We confirmed the results from the FIT system by examining endogenous cortactin in different cell types.Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain.

ABSTRACT

Background: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes.

Methodology/principal findings: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation.

Conclusions/significance: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

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Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading.(A) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. (B) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. (C) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.
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pone-0033662-g009: Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading.(A) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. (B) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. (C) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.

Mentions: We examined the spreading of HeLa cells on fibronectin, and in parallel left them in suspension as a negative control (Fig. 9). Since previous work has shown that WT cortactin interacts with FAK, while cortactin lacking the SH3 domain does not [37], we performed pull-down experiments on the lysates using recombinant purified cortactin SH3 domain fused to GST (GST-SH3) or GST alone as a negative control (Fig. 9A). We found that cortactin SH3 domain was able to pull down much more FAK from the lysates of spread cells than from lysates of suspended cells. Similar results where obtained with Rsrc lysates (data not shown). These results indicate that focal adhesion formation during cell spreading induces cortactin-FAK association (Fig. 9B).


Cortactin tyrosine phosphorylation promotes its deacetylation and inhibits cell spreading.

Meiler E, Nieto-Pelegrín E, Martinez-Quiles N - PLoS ONE (2012)

Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading.(A) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. (B) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. (C) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316595&req=5

pone-0033662-g009: Tyrosine phosphorylation of cortactin terminates its interaction with focal adhesion kinase (FAK) during cell spreading.(A) Coomassie staining of purified GST and GST-cortactin SH3 domain was scanned in the Odyssey system. (B) HeLa cells were detached with trypsin-EDTA, washed with trypsin inhibitor and kept in suspension (susp.) or allowed to spread for 3 h on fibronectin (FN)-treated 100-mm plates. RIPA cell lysates were used for pull-down experiments with GST or GST-SH3, which were analyzed by SDS-PAGE and WB with focal adhesion kinase (FAK) Ab, followed by labeling with a 800CW-conjugated goat rabbit Ab. (C) HeLa cells were transfected with ZipB-MycCortactin and empty vector (TF2) or with ZipB-MycCortactin and ZipA-HAΔSrc (TF3). After 20 h cells were detached with trypsin-EDTA, washed with trypsin inhibitor and allowed to spread on FN-coated 100-mm plates for 3 h. Cell lysates were subjected to immunoprecipitation with FAK MoAb. The immunoprecipitates were subjected to WB and probed in three steps: (1) with myc Ab to detect transfected cortactin, followed by a 680CW-labeled goat mouseAb (red); (2) with FAK Ab, followed by a 800CW-labeled goat rabbit Ab (green); and (3) with pY466 cortactin Ab, followed by a 800CW-labeled goat rabbit Ab. Transfected cortactin was immunoprecipitated by FAK (asterisk) only when the protein was not tyrosine-phosphorylated.
Mentions: We examined the spreading of HeLa cells on fibronectin, and in parallel left them in suspension as a negative control (Fig. 9). Since previous work has shown that WT cortactin interacts with FAK, while cortactin lacking the SH3 domain does not [37], we performed pull-down experiments on the lysates using recombinant purified cortactin SH3 domain fused to GST (GST-SH3) or GST alone as a negative control (Fig. 9A). We found that cortactin SH3 domain was able to pull down much more FAK from the lysates of spread cells than from lysates of suspended cells. Similar results where obtained with Rsrc lysates (data not shown). These results indicate that focal adhesion formation during cell spreading induces cortactin-FAK association (Fig. 9B).

Bottom Line: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading.We confirmed the results from the FIT system by examining endogenous cortactin in different cell types.Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain.

ABSTRACT

Background: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes.

Methodology/principal findings: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation.

Conclusions/significance: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

Show MeSH
Related in: MedlinePlus