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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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Specificity of tyrosine phosphorylation in the FIT system.(A) Detection of the phosphorylation status of paxillin, another Src kinase substrate. SYF and Rsrc cells were transfected with FIT fusion vectors and the most relevant lysates (4 and 5) from two different experiments (FIT 8 and 9) were analyzed by WB with a rabbit Ab against phospho-paxillin (in green) and with a MoAb against actin (in red). As controls, cells were left untreated or treated with pervanadate (PV), a potent phosphatase inhibitor that induces the phosphorylation of paxillin. Rsrc cells showed a higher basal level of phospho-paxillin than did SYF cells, though in both cell lines, this basal level was enhanced by treatment with PV. The FIT system did not increase the basal level of phospho-paxillin. (B) Tyrosine phosphorylation of cortactin occurs on the expected tyrosines (Y421, Y466 and Y482). HeLa cell lysates were transfected with ZipA-HA-ΔSrc and ZipB-MycCortactin (lane 4) or with ZipA-HA-ΔSrc and ZipB-MycCortactin with the triple mutation Y421/466/482F (3F) (lane 5). Several control cotransfections were done (lanes 1–3). WB with generic pTyr MoAb demonstrated that only ZipB-Myc WT cortactin, and not the 3F mutant, was phosphorylated (in green). Cortactin was detected with a rabbit MoAb (in red). Actin is shown as a loading control.
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pone-0033662-g002: Specificity of tyrosine phosphorylation in the FIT system.(A) Detection of the phosphorylation status of paxillin, another Src kinase substrate. SYF and Rsrc cells were transfected with FIT fusion vectors and the most relevant lysates (4 and 5) from two different experiments (FIT 8 and 9) were analyzed by WB with a rabbit Ab against phospho-paxillin (in green) and with a MoAb against actin (in red). As controls, cells were left untreated or treated with pervanadate (PV), a potent phosphatase inhibitor that induces the phosphorylation of paxillin. Rsrc cells showed a higher basal level of phospho-paxillin than did SYF cells, though in both cell lines, this basal level was enhanced by treatment with PV. The FIT system did not increase the basal level of phospho-paxillin. (B) Tyrosine phosphorylation of cortactin occurs on the expected tyrosines (Y421, Y466 and Y482). HeLa cell lysates were transfected with ZipA-HA-ΔSrc and ZipB-MycCortactin (lane 4) or with ZipA-HA-ΔSrc and ZipB-MycCortactin with the triple mutation Y421/466/482F (3F) (lane 5). Several control cotransfections were done (lanes 1–3). WB with generic pTyr MoAb demonstrated that only ZipB-Myc WT cortactin, and not the 3F mutant, was phosphorylated (in green). Cortactin was detected with a rabbit MoAb (in red). Actin is shown as a loading control.

Mentions: As a second control of phosphorylation specificity, we analyzed whether the Src substrate paxillin [36] is phosphorylated by our transfected Src kinase. We performed WB using a phospho-paxillin (p-paxillin) Ab. In Fig. 2A we show the most relevant transfections (lanes 4 and 5) from two FIT experiments (FIT8 and FIT9), after blotting with p-paxillin Ab. As an internal control, we treated both cell types with pervanadate, a generic phosphatase inhibitor that induced a strong signal for p-paxillin. While untreated cell lysates did not show detectable paxillin phosphorylation, lysates of treated cells did. Thus we can conclude that our transfected cells express a basal level of phospho-paxillin.


Cortactin tyrosine phosphorylation promotes its deacetylation and inhibits cell spreading.

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

Specificity of tyrosine phosphorylation in the FIT system.(A) Detection of the phosphorylation status of paxillin, another Src kinase substrate. SYF and Rsrc cells were transfected with FIT fusion vectors and the most relevant lysates (4 and 5) from two different experiments (FIT 8 and 9) were analyzed by WB with a rabbit Ab against phospho-paxillin (in green) and with a MoAb against actin (in red). As controls, cells were left untreated or treated with pervanadate (PV), a potent phosphatase inhibitor that induces the phosphorylation of paxillin. Rsrc cells showed a higher basal level of phospho-paxillin than did SYF cells, though in both cell lines, this basal level was enhanced by treatment with PV. The FIT system did not increase the basal level of phospho-paxillin. (B) Tyrosine phosphorylation of cortactin occurs on the expected tyrosines (Y421, Y466 and Y482). HeLa cell lysates were transfected with ZipA-HA-ΔSrc and ZipB-MycCortactin (lane 4) or with ZipA-HA-ΔSrc and ZipB-MycCortactin with the triple mutation Y421/466/482F (3F) (lane 5). Several control cotransfections were done (lanes 1–3). WB with generic pTyr MoAb demonstrated that only ZipB-Myc WT cortactin, and not the 3F mutant, was phosphorylated (in green). Cortactin was detected with a rabbit MoAb (in red). Actin is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316595&req=5

pone-0033662-g002: Specificity of tyrosine phosphorylation in the FIT system.(A) Detection of the phosphorylation status of paxillin, another Src kinase substrate. SYF and Rsrc cells were transfected with FIT fusion vectors and the most relevant lysates (4 and 5) from two different experiments (FIT 8 and 9) were analyzed by WB with a rabbit Ab against phospho-paxillin (in green) and with a MoAb against actin (in red). As controls, cells were left untreated or treated with pervanadate (PV), a potent phosphatase inhibitor that induces the phosphorylation of paxillin. Rsrc cells showed a higher basal level of phospho-paxillin than did SYF cells, though in both cell lines, this basal level was enhanced by treatment with PV. The FIT system did not increase the basal level of phospho-paxillin. (B) Tyrosine phosphorylation of cortactin occurs on the expected tyrosines (Y421, Y466 and Y482). HeLa cell lysates were transfected with ZipA-HA-ΔSrc and ZipB-MycCortactin (lane 4) or with ZipA-HA-ΔSrc and ZipB-MycCortactin with the triple mutation Y421/466/482F (3F) (lane 5). Several control cotransfections were done (lanes 1–3). WB with generic pTyr MoAb demonstrated that only ZipB-Myc WT cortactin, and not the 3F mutant, was phosphorylated (in green). Cortactin was detected with a rabbit MoAb (in red). Actin is shown as a loading control.
Mentions: As a second control of phosphorylation specificity, we analyzed whether the Src substrate paxillin [36] is phosphorylated by our transfected Src kinase. We performed WB using a phospho-paxillin (p-paxillin) Ab. In Fig. 2A we show the most relevant transfections (lanes 4 and 5) from two FIT experiments (FIT8 and FIT9), after blotting with p-paxillin Ab. As an internal control, we treated both cell types with pervanadate, a generic phosphatase inhibitor that induced a strong signal for p-paxillin. While untreated cell lysates did not show detectable paxillin phosphorylation, lysates of treated cells did. Thus we can conclude that our transfected cells express a basal level of phospho-paxillin.

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