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Identification of two tyrosine residues required for the intramolecular mechanism implicated in GIT1 activation.

Totaro A, Astro V, Tonoli D, de Curtis I - PLoS ONE (2014)

Bottom Line: Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX.Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin.Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin.

View Article: PubMed Central - PubMed

Affiliation: Cell Adhesion Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be sufficient to release the intramolecular interaction that keeps GIT1 in the inactive conformation.

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Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.(A) Schematic representation of human GIT1 (NP 001078923.1). GFP-tagged wild type GIT1 was used to introduce phosphomimetic mutations. Tyrosine (Y) or serine (S) and threonine (T) residues were mutated into glutamic acid or aspartic acid, respectively. White and black stars indicate the locations of the mutated tyrosine and serine/threonine residues, respectively. ArfGAP, Arf GTPase-activating protein; ANK, ankyrin repeats; SHD, Spa2-homology domain; LZ, leucine zipper; PBS, paxillin binding site; SLD, synaptic localization domain. (B) Aliquots of lysates (400 μg) from cells transfected with the indicated constructs were used for immunoprecipitation of endogenous paxillin. Filters with immunoprecipitates (IP), and equal amounts (80 μg) of the respective lysates (Ly) or unbound fractions after immunoprecipitation (Ub) were blotted with anti-GFP (for GIT1) or anti-paxillin antibodies. Molecular weight markers are indicated to the right of each blot. (C) The double substitution of residues Y246 and Y293 with either two glutamic acid (EE) or two alanine residues (AA) enhanced GIT1 binding to paxillin. Aliquots of lysates (200 μg protein) from cells transfected with the indicated constructs were immunoprecipitated with anti-paxillin antibody. Filters with immunoprecipitates (IP), and lysates (40 μg) were blotted as indicated, using anti-GFP (for GIT1) or anti-paxillin antibodies. (D) Model for GIT1 activation: see details in the text.
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pone-0093199-g001: Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.(A) Schematic representation of human GIT1 (NP 001078923.1). GFP-tagged wild type GIT1 was used to introduce phosphomimetic mutations. Tyrosine (Y) or serine (S) and threonine (T) residues were mutated into glutamic acid or aspartic acid, respectively. White and black stars indicate the locations of the mutated tyrosine and serine/threonine residues, respectively. ArfGAP, Arf GTPase-activating protein; ANK, ankyrin repeats; SHD, Spa2-homology domain; LZ, leucine zipper; PBS, paxillin binding site; SLD, synaptic localization domain. (B) Aliquots of lysates (400 μg) from cells transfected with the indicated constructs were used for immunoprecipitation of endogenous paxillin. Filters with immunoprecipitates (IP), and equal amounts (80 μg) of the respective lysates (Ly) or unbound fractions after immunoprecipitation (Ub) were blotted with anti-GFP (for GIT1) or anti-paxillin antibodies. Molecular weight markers are indicated to the right of each blot. (C) The double substitution of residues Y246 and Y293 with either two glutamic acid (EE) or two alanine residues (AA) enhanced GIT1 binding to paxillin. Aliquots of lysates (200 μg protein) from cells transfected with the indicated constructs were immunoprecipitated with anti-paxillin antibody. Filters with immunoprecipitates (IP), and lysates (40 μg) were blotted as indicated, using anti-GFP (for GIT1) or anti-paxillin antibodies. (D) Model for GIT1 activation: see details in the text.

Mentions: We have previously shown that either endogenous or overexpressed GIT1 binds very poorly to endogenous paxillin, while deletion of different segments of the amino-terminal portions of the polypeptide induces strong binding of paxillin to the carboxy-terminal portion of the truncated proteins [23]. To limit the possible effects of these deletions on the overall structure of the GIT1 protein, we have tested here the hypothesis that activation may be obtained by site-directed mutagenesis of specific residues of GIT1. A number of studies indicate that GIT1 is phosphorylated in response to different stimuli [7], [24]–[26], and that phosphorylation may modulate GIT1 functions in different cells [14], [27]–[28]. For this reason, we have considered several residues that have been shown to be sites of phosphorylation on the GIT1 proteins (Figure 1A, Figure S1) [29], and tested whether one or more of these sites were able to regulate GIT1 function measured as its ability to bind paxillin.


Identification of two tyrosine residues required for the intramolecular mechanism implicated in GIT1 activation.

Totaro A, Astro V, Tonoli D, de Curtis I - PLoS ONE (2014)

Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.(A) Schematic representation of human GIT1 (NP 001078923.1). GFP-tagged wild type GIT1 was used to introduce phosphomimetic mutations. Tyrosine (Y) or serine (S) and threonine (T) residues were mutated into glutamic acid or aspartic acid, respectively. White and black stars indicate the locations of the mutated tyrosine and serine/threonine residues, respectively. ArfGAP, Arf GTPase-activating protein; ANK, ankyrin repeats; SHD, Spa2-homology domain; LZ, leucine zipper; PBS, paxillin binding site; SLD, synaptic localization domain. (B) Aliquots of lysates (400 μg) from cells transfected with the indicated constructs were used for immunoprecipitation of endogenous paxillin. Filters with immunoprecipitates (IP), and equal amounts (80 μg) of the respective lysates (Ly) or unbound fractions after immunoprecipitation (Ub) were blotted with anti-GFP (for GIT1) or anti-paxillin antibodies. Molecular weight markers are indicated to the right of each blot. (C) The double substitution of residues Y246 and Y293 with either two glutamic acid (EE) or two alanine residues (AA) enhanced GIT1 binding to paxillin. Aliquots of lysates (200 μg protein) from cells transfected with the indicated constructs were immunoprecipitated with anti-paxillin antibody. Filters with immunoprecipitates (IP), and lysates (40 μg) were blotted as indicated, using anti-GFP (for GIT1) or anti-paxillin antibodies. (D) Model for GIT1 activation: see details in the text.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3974724&req=5

pone-0093199-g001: Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.(A) Schematic representation of human GIT1 (NP 001078923.1). GFP-tagged wild type GIT1 was used to introduce phosphomimetic mutations. Tyrosine (Y) or serine (S) and threonine (T) residues were mutated into glutamic acid or aspartic acid, respectively. White and black stars indicate the locations of the mutated tyrosine and serine/threonine residues, respectively. ArfGAP, Arf GTPase-activating protein; ANK, ankyrin repeats; SHD, Spa2-homology domain; LZ, leucine zipper; PBS, paxillin binding site; SLD, synaptic localization domain. (B) Aliquots of lysates (400 μg) from cells transfected with the indicated constructs were used for immunoprecipitation of endogenous paxillin. Filters with immunoprecipitates (IP), and equal amounts (80 μg) of the respective lysates (Ly) or unbound fractions after immunoprecipitation (Ub) were blotted with anti-GFP (for GIT1) or anti-paxillin antibodies. Molecular weight markers are indicated to the right of each blot. (C) The double substitution of residues Y246 and Y293 with either two glutamic acid (EE) or two alanine residues (AA) enhanced GIT1 binding to paxillin. Aliquots of lysates (200 μg protein) from cells transfected with the indicated constructs were immunoprecipitated with anti-paxillin antibody. Filters with immunoprecipitates (IP), and lysates (40 μg) were blotted as indicated, using anti-GFP (for GIT1) or anti-paxillin antibodies. (D) Model for GIT1 activation: see details in the text.
Mentions: We have previously shown that either endogenous or overexpressed GIT1 binds very poorly to endogenous paxillin, while deletion of different segments of the amino-terminal portions of the polypeptide induces strong binding of paxillin to the carboxy-terminal portion of the truncated proteins [23]. To limit the possible effects of these deletions on the overall structure of the GIT1 protein, we have tested here the hypothesis that activation may be obtained by site-directed mutagenesis of specific residues of GIT1. A number of studies indicate that GIT1 is phosphorylated in response to different stimuli [7], [24]–[26], and that phosphorylation may modulate GIT1 functions in different cells [14], [27]–[28]. For this reason, we have considered several residues that have been shown to be sites of phosphorylation on the GIT1 proteins (Figure 1A, Figure S1) [29], and tested whether one or more of these sites were able to regulate GIT1 function measured as its ability to bind paxillin.

Bottom Line: Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX.Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin.Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin.

View Article: PubMed Central - PubMed

Affiliation: Cell Adhesion Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be sufficient to release the intramolecular interaction that keeps GIT1 in the inactive conformation.

Show MeSH
Related in: MedlinePlus