Hyperphosphorylation of GIT1-N by Src and pervanadate does not affect its binding <i>in vitro</i> to full length GIT1 proteins.

<p>(<b>A–C</b>) COS7 cells were transfected with full length GFP-GIT1 or GIT1-N constructs (WT, FF, or EE), or with wildtype or mutant FLAG-GIT1-N fragments, alone or together with c-Src. The cells cotransfected with c-Src were incubated 20 min at 37°C with 1 mM pervanadate before lysis (pV). Aliquots of the lysates (200 μg of protein) were immunoprecipitated (IP) with anti-FLAG (<b>A,C</b>) or anti-GFP (<b>B</b>) antibodies. For pulldowns shown in (<b>A</b>) and (<b>C</b>): FLAG-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (250 μg of protein) from cells transfected with the indicated full length GFP-GIT1 constructs. Equal amounts of lysates (25 μg of protein), and the pulldowns performed with GIT1-N without (<b>A</b>) or with c-Src and pervanadate treatment (<b>C</b>) were blotted for the detection of the indicated antigens. In (<b>B</b>) the immunoprecipitations with anti-GFP antibody (right) were immunoblotted to detect the levels of GFP-GIT1-N protein (upper filter) and of its tyrosine phosphorylation (lower filter). (<b>D</b>) COS7 cells were transfected to express the indicated GFP-GIT1 mutants, or transfected with the HA-GIT1-N fragment alone or together with c-Src. The cells co-transfected with c-Src were treated as in (<b>A,C</b>). Aliquots of the lysates from cells expressing GFP-GIT1 mutants (250 μg of protein) were immunoprecipitated with anti-GFP. Pulldowns: GFP-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (400 μg of protein) from cells transfected with HA-GIT1-N alone, or together with c-Src. Equal amounts of lysates (25 μg of protein), and the pulldowns were blotted for the detection of the indicated antigens.</p

Efficient binding of the amino-terminal portion of GIT1 to the carboxy-terminal portion requires the inclusion of the first Spa2 region of the SHD.

<p>(<b>A</b>) Scheme of the constructs of avian GIT1 used in the following experiment. To be noted that tyrosine 284 of avian GIT1 corresponds to tyrosine 293 of human GIT1. (<b>B</b>) COS7 cells were cotransfected with the indicated combination of HA-tagged amino-terminal and FLAG-tagged carboxy-terminal fragments of GIT1. Lysates were immunoprecipitated with the anti-FLAG M2-conjugated beads. Filters with immunoprecipitates (IP, from 250 μg of protein lysate) and lysates (50 μg) were incubated with anti-HA or anti-FLAG antibodies. Amino-terminal fragments in the lysates are indicated by asterisks.</p

Mutation of tyrosines 246 and 293 does not affect the binding of GIT1 to βPIX.

<p>(<b>A</b>) Lysates from mock-transfected cells or from cells transfected with the indicated GIT1 constructs were immunoprecipitated for GIT1 with anti-GFP antibody. Filters with immunoprecipitates (IP, from 200 μg of protein lysate), and lysates (25 μg) were blotted with anti-GFP (for GIT1), anti-paxillin, and anti-βPIX antibodies. (<b>B</b>) Binding of GIT1-N to GIT1-Y246E/Y293E does not affect the interaction of GIT1-Y246E/Y293E to βPIX. Lysates from COS7 cells co-transfected to express the HA-GIT1-N fragment and the full length GFP-GIT1-WT or GIT1-Y246E/Y293E proteins were immunoprecipitated (IP, from 175 μg of protein lysate) with anti-GFP. Lysates (25 μg, oƒn the lef) and IP (right) were blotted to reveal the full length proteins (anti-GFP), the HA-GIT1-N fragment (anti-HA mAb 12CA5), and endogenous βPIX. (<b>C</b>) Our data support the hypothesis that the tyrosine 293 of the SHD domain of GIT1 is required for the intramolecular interaction, but not for the interaction of the SHD domain of GIT1 with βPIX.</p

Effects of tyrosine phosphorylation on the binding of GIT1 to paxillin.

<p>COS7 cells were transfected with GIT1 constructs (<b>A</b>) or cotransfected with the GIT1 constructs together with c-Src (<b>B</b>). The cells cotransfected with c-Src were also treated for 20 min at 37°C with 1 mM pervanadate before lysis (pV). In parallel, a lysate was prepared from cells transfected with orange-paxillin (PXN). After immunoprecipitation of each GFP-GIT1 construct with anti-GFP antibodies (250 μg of protein lysate/immunoprecipitation), the beads were incubated for 2 h at 4°C with aliquots of the lysate containing orange-paxillin (180 μg). Lysates (25 μg) and immunoprecipitates (IP) after pulldown were used for immunoblotting for the indicated antigens.</p

Mutations Y246E and Y293E of GIT1 enhance binding to paxillin.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) Model for GIT1 activation: see details in the text.</p

Expression of the “active” GIT1-Y246E/Y293E mutant increases the efficiency of migration.

<p>(<b>A</b>) COS7 cells transfected with GFP or with GFP-tagged GIT1 constructs were used for haptotactic cell migration towards fibronectin. Images show fields with cells migrated to the lower side of the filters coated with fibronectin. (<b>B,C</b>) Quantification of the number of cells migrated to the lower side of the wells. Bars are normalized mean values ±SEM from 12–19 fields from 2–3 independent experiments. **P<0.02. (<b>D–F</b>) Wound-healing assay. COS7 cells were transfected as in (<b>A</b>). Confluent monolayers were wounded and followed by time lapse imaging. (<b>D</b>) Fluorescent images (left column) showing GFP-positive transfected COS7 cells at time 0. The other columns show the phase contrast images of the same fields, at the beginning (t = 0) and the end (t = 6 h) of the assay. Bar, 100 μm. (<b>E</b>) Tracks of GFP-positive cells transfected with either GFP-GIT1 or GFP-GIT1-Y246E/Y293E. Tracks were taken during the wound healing assays and refer to an interval of 8 h. (<b>F</b>) Quantification of the euclidean distance covered by cells during the wound healing assay. Bars are mean values ±SEM from 90 cells from 3 different experiments. *P<0.05; ***P<0,002.</p

Tyrosines 246 and 293 are required to hold GIT1 in a closed conformation.

<p>(<b>A,C,D,F</b>) COS7 cells were cotransfected with the indicated constructs, lysed, and immunoprecipitated with anti-GFP (<b>A,C,F</b>) or anti-FLAG antibodies (<b>D</b>). Filters with immunoprecipitates (IP) and lysates were blotted to reveal the full length GFP-tagged constructs (with anti-GFP), HA-GIT1-N (with mouse 12CA5 mAb), or GIT1-C2-LZ fragment (with anti-FLAG). (<b>B,E,G</b>) Schemes of the interactions proposed from the experiments presented in (<b>A</b>), (<b>C,D</b>), and (<b>F</b>), respectively. The asterisks in (<b>E</b>) indicate the monomeric mutated carboxy-terminal fragment that can not dimerize. See the Results for details.</p

Identification of Two Tyrosine Residues Required for the Intramolecular Mechanism Implicated in GIT1 Activation

<div><p>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.</p></div