Beta-tubulin binds Src homology 2 domains through a region different from the tyrosine-phosphorylated protein-recognizing site.

Src homology 2 (SH2) domains have been demonstrated to bind tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes by recognizing amino acid sequences containing phosphotyrosine residue. We found that SH2 domains such as Ash/Grb2, the 85-kDa subunit of phosphatidylinositol 3-kinase, and phospholipase Cγ1 also bind β-tubulin through a different region that recognizes phosphotyrosine in vitro and in vivo. Furthermore, binding occurs even when the SH2 domain is occupied by tyrosine-phosphorylated epidermal growth factor receptors. Using deleted constructs of Ash/Grb2 SH2, we found that carboxyl-terminal β strands E and F, and α helix B (region “c”) are required for binding. A synthetic peptide (FLWVVKFNSLNELVDYH) composed of region c inhibited the binding of β-tubulin to the SH2 domains of Ash/Grb2, phosphatidylinositol 3-kinase, and phospholipase Cγ1. The co-localization of SH2 proteins and microtubules is also confirmed by immunostaining. These data suggest that microtubules play important roles in the assembly of signaling molecules complexes containing SH2 proteins.

strated to bind tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes by recognizing amino acid sequences containing phosphotyrosine residue. We found that SH2 domains such as Ash/Grb2, the 85-kDa subunit of phosphatidylinositol 3-kinase, and phospholipase C␥1 also bind ␤-tubulin through a different region that recognizes phosphotyrosine in vitro and in vivo. Furthermore, binding occurs even when the SH2 domain is occupied by tyrosine-phosphorylated epidermal growth factor receptors. Using deleted constructs of Ash/Grb2 SH2, we found that carboxyl-terminal ␤ strands E and F, and ␣ helix B (region "c") are required for binding. A synthetic peptide (FLWVVKFNSLNELVDYH) composed of region c inhibited the binding of ␤-tubulin to the SH2 domains of Ash/Grb2, phosphatidylinositol 3-kinase, and phospholipase C␥1. The co-localization of SH2 proteins and microtubules is also confirmed by immunostaining. These data suggest that microtubules play important roles in the assembly of signaling molecules complexes containing SH2 proteins.
Src homology 2 and 3 (SH2 and SH3) domains are independent modular units found in a variety of proteins involved in signal transduction (1)(2)(3)(4)(5). SH2 domains are amino acid sequences that are similar to a 100-residue noncatalytic region of the Src tyrosine kinase and contain binding sites for phosphotyrosine. The interaction between SH2 domains and tyrosinephosphorylated proteins is important in the assembly of signal transduction complexes. Besides SH2 domains, many proteins regulated by tyrosine kinases have SH3 domains, which recognize proline-rich sequences of downstream signaling molecules. SH3 domains have also been identified in several cytoskeletal proteins, including spectrin, myosin I, and an actin-binding protein from yeast, ABP-1 (6 -8), suggesting that these domains are involved in regulating the interaction of signaling molecules with the cytoskeleton. In addition, SH2 domain-and SH3 domain-containing molecules, such as PLC␥, 1 PI 3-kinase, and p60 c-src , have been shown to be localized in the cytoskeleton, and SH3 domains have been implicated as important for localization in the cytoskeleton (9 -12). However, the relationship between SH2/SH3 domains and the cytoskeleton remains unclear.
Ash/Grb2, composed entirely of SH2 and SH3 domains, was found independently by two groups (13,14) and demonstrated to bind to autophosphorylated EGF receptors and other phosphotyrosine-containing proteins such as Shc and insulin receptor substrate 1 through the SH2 domain (15)(16)(17)(18)(19)(20). Ash/Grb2 also forms complexes with Sos, a mammalian homolog of the Drosophila guanine-nucleotide-releasing factors for Ras (21), through the SH3 domain, suggesting that Ash/Grb2 is involved in Ras activation signaling. This is likely to be due to an interaction between the SH3 of Ash/Grb2 and a proline-rich sequence in Sos.
A recent study has shown that the injection of cells with an antibody against Ash/Grb2 abolishes the reorganization of actin stress fibers (22). In addition, it has been found that dynamin binds to Ash/Grb2 through SH3 domains (23) and that its GTPase activity is stimulated by binding to Ash/Grb2 (24). These findings suggest that Ash/Grb2 functions not only in Ras signaling but also in other pathways.
Here, we report that ␤-tubulin binds SH2 domains through a region different from the tyrosine-phosphorylated protein-recognizing site. We determined that carboxyl-terminal ␤ strands E and F and ␣ helix B form the binding site of ␤-tubulin.
Synthetic peptides corresponding to the sequence of the Ash/Grb2binding site of the autophosphorylated EGF receptor (PVPEpYINQS-VPK) were purchased from Peptide Institute Inc. (Osaka, Japan). The Ash/Grb2 SH2 "c" region (FLWVVKFNSLNELVDYH) and control peptides (FLWVVKFNSLN and CPPCPALWLK) were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) method with a peptide synthesizer (Applied Biosystems). The termini of all peptides were deblocked.
Identification of a 55-kDa Protein Bound to GST-Ash/Grb2 as ␤-Tubulin-Recombinant GST-Ash fusion protein (23) coupled to glutathione-Sepharose was used to affinity purify Ash-binding proteins from the cytosolic fractions of bovine brains. The bound proteins were eluted with 20 mM Tris-HCl (pH 7.4) containing 50 mM glutathione, subjected to SDS-polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride membranes. Protein bands at 55 kDa were cut out, digested with lysylendopeptidase, and separated on a C 18 reverse-phase column. We sequenced two peptides from the digested 55-kDa protein and found them (MREIVHIQAG, IREEYPDRIMNTFSVMP) to be identical with the partial sequence of bovine ␤-tubulin.
GST Fusion Proteins-Bacterial expression plasmids coding GST fusion proteins were produced by in-frame insertion of fragments corresponding to each region. GST-PLC␥1 SH2 expression plasmid was made by cutting the cDNA of rat PLC␥1 with XhoI and PvuII, ligating it to EcoRI linker, and inserting it into the EcoRI site of pGEX-3X. GST-p85 SH2 expression plasmid, which includes both N and CSH2 domains, was made by cutting the cDNA of human p85 with SnaI and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Purification of Microtubule Protein and the Association of Tubulin with SH2 Proteins in Vivo-Bovine brain was homogenized in reassembly buffer (RB) containing 1 mM GTP and centrifuged at 20,000 ϫ g. To the supernatant were added one-third volume of glycerol and GTP (1 mM final). The mixtures were then warmed at 37°C to polymerize and were centrifuged at 100,000 ϫ g. The pellet was suspended in cold RB, homogenized, incubated at 4°C to depolymerize, and centrifuged at 100,000 ϫ g. This polymerization-depolymerization cycle was repeated once more, and microtubule proteins were obtained in the depolymerized form in the supernatant. Co-polymerization was analyzed by further polymerization-depolymerization cycles starting with crude microtubule proteins obtained as described above. The crude microtubule proteins were polymerized and centrifuged to yield supernatant 1 and precipitate 1. Then, precipitate 1 was suspended in cold RB, sonicated briefly, cooled to depolymerize, and centrifuged to yield supernatant 2 and precipitate 2. Finally, supernatant 2 was polymerized and centrifuged to yield supernatant 3 and precipitate 3. Each supernatant and pellet (suspended in a volume of RB equal to that of the supernatants) was separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted with antibodies against each SH2-containing protein.
For further purification of tubulin protein from other microtubule proteins such as microtubule-associated proteins or tau, the crude microtubule proteins were loaded into a P11 phosphocellulose column. Tubulin was obtained in the flow-through fractions.
Kinetics of the Interaction between SH2 Domains and ␤-Tubulin-Tubulin purified from bovine brain was labeled with 125 I using N-succinimidyl-3-(4-hydroxy-3-[ 125 I]iodophenyl)propionate (Bolton-Hunter reagent). Various concentrations of labeled tubulin were subjected to SH2 binding. After washing, bound tubulin was counted in a gamma counter, and the K d value of the interaction was estimated by Scatchard plot.
Inhibition of ␤-Tubulin Binding by Synthetic Peptides-Various concentrations of a peptide (FLWVVKFNSLNELVDYH) were preincubated for 2 h with bovine brain cytosol fractions (5 mg of total protein); then immobilized GST-Ash/Grb2 SH2 domain was added. Bound ␤-tubulin was detected by immunoblotting with anti-␤-tubulin antibody.
Immunofluorescent Staining-Swiss 3T3 mouse fibroblasts were grown on coverslips in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were fixed with methanol at Ϫ20°C for 6 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 1 min. After blocking with phosphate-buffered saline containing 10% calf serum and 3% BSA, incubation with the first and second antibodies was performed for 1 h each. The coverslips were then washed FIG. 1. SH2 domains specifically bind to ␤-tubulin in vitro. A, the SH2 domain, not SH3 domains, of Ash/Grb2 is responsible for the binding to ␤-tubulin. GST fusion proteins were immobilized to beads and incubated with bovine brain cytosol fractions. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted (I.B.) with anti-␤-tubulin antibody. B, PLC␥1 and PI 3-kinase 85-kDa subunit SH2 domains can also bind ␤-tubulin. GST-SH2 domains of p85 and PLC␥1 were subjected to the ␤-tubulin binding assay as in A. C, direct interaction between tubulin and SH2 domains. Tubulin protein (20 g) purified from bovine brain was mixed with various GST-SH2 constructs (50 g) immobilized to beads. Bound tubulin was visualized with anti-␤-tubulin antibody.
FIG. 2. Co-immunoprecipitation of ␤-tubulin with Ash/Grb2, PI 3-kinase, and PLC␥1. A, immunoprecipitation analysis. Ash/Grb2, p85, and PLC␥1 were immunoprecipitated (I.P.) with polyclonal antibody against each protein, and the precipitates were immunoblotted (I.B.) with monoclonal anti-␤-tubulin antibody. B, co-polymerization assay. Microtubule proteins, including tubulins and microtubule-associated proteins, were obtained from bovine brain. The crude tubulin proteins were subjected to polymerizing-depolymerizing cycles and the SH2-containing proteins in the supernatant or pellet at each step were detected by Western blotting. sup, supernatant; ppt, precipitate. and mounted on slides with 1 mg/ml p-phenylenediamine, 90% glycerol in phosphate-buffered saline.
Localization of FITC-labeled GST-SH2 c in Microtubules-The colocalization of Ash/Grb2 SH2 c to microtubules was investigated by incubating cells with FITC-labeled GST-SH2 c in microtubule-stabilizing buffer containing 0.2% Triton X-100 (29) to permeabilize the membranes. GST-Ash SH2 c was labeled with FITC (Polyscience, Inc.) and separated from free FITC by gel filtration. The extent of labeling was checked by SDS-polyacrylamide gel electrophoresis.

RESULTS
Identification of an Ash/Grb2-binding Protein as ␤-Tubulin-To obtain Ash/Grb2-binding proteins, we expressed Ash/ Grb2 as a GST fusion protein in Escherichia coli, immobilized it to glutathione-Sepharose, and carried out affinity purification. The cytosol fractions of bovine brain were applied to the GST-Ash/Grb2 affinity column, and several Ash/Grb2 binding proteins were obtained (23). Among them, a 55-kDa protein was digested by lysylendopeptidase, and the peptides were separated on a C 18 reverse-phase column. The amino acid sequences of two peptides were determined (MREIVHIQAG and IREEYPDRIMNTFSVMP) and found to be identical with sequences in ␤-tubulin. Western blotting analysis with antibody against ␤-tubulin also confirmed that the 55-kDa protein is ␤-tubulin (Fig. 1A, lane 2).
␤-Tubulin Binds to SH2 Domains of Ash/Grb2, PI 3-kinase, and PLC␥1-It is believed that Ash/Grb2 binds to its binding proteins via the SH2 or the SH3 domain because Ash/Grb2 is composed of only SH2 and SH3 domains. However, ␤-tubulin is not tyrosine phosphorylated (data not shown) and has no proline-rich motif that is likely to bind to SH3 domains. Thus, it is of great interest to identify the ␤-tubulin-binding domain within Ash/Grb2. To clarify which domain is responsible for the association, we used GST fusion proteins containing each do-main of Ash/Grb2 and found that the SH2 domain could bind strongly to ␤-tubulin (Fig. 1A). SH2 domains of the 85-kDa subunit of PI 3-kinase (p85) and PLC␥1 could also bind to ␤-tubulin (Fig. 1B). This indicates that association with ␤-tubulin is a common characteristic conserved among SH2 domains.
To clarify whether the interaction between SH2 domains and ␤-tubulin is direct or not, tubulin proteins were purified from bovine brain and subjected to binding assay with SH2 domains. While ␤-tubulin did not bind to GST alone, a significant amount of ␤-tubulin was shown to bind to all SH2 domains tested (Fig. 1C).
The kinetics of the interaction between ␤-tubulin and SH2 domain was investigated Scatchard analysis using 125 I-labeled tubulin protein, and the K d was estimated to be 4.2 M.
Ash/Grb2, PI 3-Kinase, and PLC␥1 Co-polymerize and Codepolymerize with Microtubules-We purified microtubule protein, which includes tubulins and microtubule-associated proteins, from bovine brain by two-cycle polymerizationdepolymerization steps. The microtubule protein obtained was further polymerized and depolymerized by heating and cooling; then the coexistence of SH2-containing proteins and tubulin was examined by immunoblotting (Fig. 2B). A portion of Ash/ Grb2, p85, and PLC␥1 coexisted with tubulin in precipitate 1, supernatant 2, and precipitate 3, which all included large amounts of tubulin (Fig. 2B). This indicates that Ash/Grb2, GST-Ash/Grb2 SH2 immobilized to beads was incubated with increasing amounts (0, 10, and 1000 g) of tubulin purified from bovine brain and then incubated with membrane fractions from A431 cells containing autophosphorylated EGF receptor. Bound EGF receptors were detected by Western blotting with anti-EGF receptor antibody (top). GST-Ash/Grb2 SH2 was incubated with membrane fractions from A431 cells containing autophosphorylated EGF receptor (0, 100, and 1000 g protein) prior to incubation with purified tubulin protein.
Bound ␤-tubulin was detected by Western blotting (bottom). B, an immobilized phosphopeptide corresponding to the Ash/Grb2-binding site of the EGF receptor can precipitate ␤-tubulin via Ash/Grb2. A phosphopeptide corresponding to the Ash SH2-binding site of the autophosphorylated EGF receptor was immobilized to CNBr-Sepharose. Ash/Grb2 was affinity purified with this phosphopeptide column via its SH2 domain. Ash/Grb2 and its binding proteins were stained with Coomassie Brilliant Blue (CBB), and the co-precipitated ␤-tubulin was detected by immunoblotting (I.B.). Ash/Grb2-binding proteins A, B, C, D, and E were revealed to be Sos, synaptojanin, c-Cbl, dynamin, and unknown protein, respectively, by partial amino acid sequences and Western blotting (data not shown).  -150; ␤G). B, binding assay using various deletion constructs. Bovine brain cytosol fraction (10 mg of protein) was incubated with 50 g of each deletion construct immobilized to beads. ␤-Tubulin bound to each Ash/Grb2 SH2 deletion construct was detected by Western blotting with monoclonal anti-␤-tubulin antibody. I.B., immunoblotting.
p85, and PLC␥1 associate tightly with microtubules despite polymerization and depolymerization.
SH2 Domains Can Bind to ␤-Tubulin and Tyrosine-phosphorylated Proteins Simultaneously-SH2 domains are known to recognize amino acid sequences containing a phosphotyrosine residue. However, ␤-tubulin, which binds to the SH2 domain of Ash/Grb2, is not tyrosine phosphorylated (data not shown). To determine whether the association between the SH2 domain and ␤-tubulin is distinct from that between SH2 domains and tyrosine-phosphorylated proteins, we investigated whether ␤-tubulin can inhibit the association between SH2 domains and tyrosine-phosphorylated proteins and vice versa. GST-SH2 of Ash/Grb2 immobilized to glutathione-Sepharose was preincubated with purified tubulin and then incubated with membrane fractions from A431 cells, which contain autophosphorylated EGF receptors. The bound EGF receptor was detected by immunoblotting with anti-EGF receptor antibody. The SH2 domain efficiently precipitated EGF receptor regardless of the presence of bound ␤-tubulin and could precipitate ␤-tubulin when already associated with tyrosine-phosphorylated EGF receptor (Fig. 3A). Furthermore, we precipitated Ash/Grb2 from bovine brain with an immobilized peptide corresponding to the Ash/Grb2-binding motif in the autophosphorylated EGF receptor. The Ash/Grb2 bound to peptide still could associate with ␤-tubulin (Fig. 3B).
A Carboxyl-terminal Region of the SH2 Domain Is Responsible for Binding to ␤-Tubulin-To identify the ␤-tubulin binding site within the Ash/Grb2 SH2 domain, we constructed a series of GST-SH2 deletion mutants for tubulin binding assays (Fig.  4A). The Ash SH2 domain was divided into four regions, designated a, b, c, and d, and mutants lacking different regions were expressed as GST fusion proteins (Fig. 4A) and used for ␤-tubulin binding assays. GST-bcd and GST-cd, which do not contain the conserved arginine residue known to interact with phosphate in phosphotyrosine, still precipitated ␤-tubulin (Fig.  4B); however, GST-d did not. Finally, we found that GST-c was sufficient to precipitate ␤-tubulin and concluded that the SH2 c region is the ␤-tubulin-binding site.
To confirm that the c region is necessary for tubulin binding, we used a synthetic peptide (FLWVVKFNSLNELVDYH) corresponding to the sequence of the c region as a competitor of the Ash SH2 domain binding to ␤-tubulin. Increasing concentrations of the peptide were preincubated with bovine brain cytosol fractions, and then immobilized GST-Ash SH2 was added. Bound ␤-tubulin was immunoblotted with anti-␤-tubulin antibody. Peptide c inhibited the association in a concentration-dependent manner (Fig. 5). We also tested the ability of peptide c to inhibit the binding of other SH2 domains to ␤-tubulin and found that it also inhibits the SH2 domains of p85 and PLC␥1 (Fig. 5). This indicates that the c region (including ␤ strands E and F and ␣ helix B) is necessary for the association. However, a peptide corresponding to the last 11 residues (FNSL-NELVDYH) within the c region (which is especially conserved among SH2 domains and can be boxed within c) did not inhibit binding. Next, to test the possibility that the binding between ␤-tubulin and SH2 domains occurred via hydrophobic interaction, peptides corresponding to the hydrophobic sequence within the former half of region c or a hydrophobic sequence within an unrelated protein, PLC␦1, were used for binding inhibition assay. In contrast to peptide c, these peptides did not inhibit the binding at any concentration.
Co-localization of SH2-containing Proteins with Microtubules in Vivo-To confirm the coexistence of Ash/Grb2, p85, or PLC␥1 and ␤-tubulin, Swiss 3T3 cells were immunostained with each antibody. Fig. 6, A-C, clearly shows that a part of the Ash/Grb2, p85, and PLC␥1 co-localizes in microtubules.
Furthermore, to examine whether SH2 c recognizes microtubules in permeabilized Swiss 3T3 cells, fluorescent-labeled GST-SH2 c was incubated with Swiss 3T3 cells in the presence of 0.2% Triton X-100. Fig. 6D shows that GST-SH2 c localizes in microtubules, suggesting that SH2 c is the tubulin-binding site. DISCUSSION We have demonstrated that ␤-tubulin binds to the SH2 domains of Ash/Grb2, PI 3-kinase, and phospholipase C␥1. This association is direct as shown by the binding of ␤-tubulin to GST-SH2 fusion proteins. Furthermore, we determined that strands E and F and ␣ helix B in the SH2 domain are required for binding. The crystal structure and nuclear magnetic resonance structure of SH2 domains revealed that the core element is an antiparallel ␤ sheet sandwiched between two ␣ helices. The central ␤ sheet (strands B, C, and D) is at the core of the structure and divides the domain into two functionally distinct regions (30 -33). One site, containing ␣ helix A and one face of the central sheet, is concerned primarily with phosphotyrosine binding. The other site (strands E, F and helix B) provides binding sites for the peptide residues immediately following the phosphotyrosine. ␤-Tubulin binds to SH2 domains even after the SH2 domains are occupied by tyrosine-phosphorylated proteins. This indicates that strands E and F and helix B, although important for recognizing the peptide sequence following a phosphotyrosine residue, can also bind ␤-tubulin simultaneously. This region is rich in hydrophobic amino acids, FIG. 5. A synthetic peptide inhibits the binding of ␤-tubulin to Ash/Grb2, PI 3-kinase, and PLC␥1 SH2 proteins. A, a synthetic peptide corresponding to Ash/Grb2 SH2 c region competitively inhibits the association of SH2 domains and ␤-tubulin. Bovine brain cytosol fractions (5 mg of protein) were preincubated with a peptide corresponding to the ␤-tubulinbinding site, GST-SH2s were added, and the mixtures were incubated. Bound ␤-tubulin was detected by Western blotting with anti-␤-tubulin. B, same inhibition assay using peptides corresponding to the hydrophobic region of peptide c or an unrelated protein, PLC␦1. which may be important for binding ␤-tubulin. We also demonstrated that a synthetic peptide (FLWVVKFNSLNELVDYH) corresponding to region c (Fig. 4A) inhibits the binding of ␤-tubulin. Furthermore, the peptide inhibits the binding of ␤-tubulin not only to Ash/Grb2 SH2 but also to PI 3-kinase and phospholipase C␥ SH2, suggesting that the binding of ␤-tubulin to SH2 domains is a common characteristic of a variety of SH2 domains. However, a synthetic peptide (FNSLNELVDYH) composed of 11 amino acids within ␣ helix B does not inhibit binding although ␣ helix B is a highly conserved area; strands E and F may be more important for ␤-tubulin binding than ␣ helix B.
Tubulin is a heterodimer composed of ␣and ␤-tubulin subunits. These subunits polymerize to generate microtubules that provide a cytoskeletal network and also associate with various proteins designated microtubule-associated proteins. Kapeller et al. (34) showed that PI 3-kinase localizes in microtubules and moves to the perinuclear area when cells are stimulated by platelet-derived growth factor, suggesting that platelet-derived growth factor receptor-PI 3-kinase complexes internalize and transit in association with the microtubule cytoskeleton. More recently, the same authors (35) reported that tubulin binds to the inter-SH2 domain of PI 3-kinase between the amino-terminal and carboxyl-terminal SH2 domains, but not to a SH2 domain. However, they did not succeed in determining the precise site for ␤-tubulin binding. We here demonstrate that GST-Ash/Grb2 SH2 and a synthetic peptide corresponding to ␤ strands E and F and ␣ helix B of Ash/Grb2 SH2 also inhibit the binding of ␤-tubulin to SH2 domains of PI 3-kinase and PLC␥1. Moreover, we found that the synthetic peptide alone could bind to ␤-tubulin (data not shown). Therefore, it is likely that the c region is sufficient for binding.
In the tyrosine kinase signaling system, tubulin may play important roles in the receptor-induced endocytosis of signaling molecules through binding to SH2-containing proteins. It is also possible that microtubules regulate the assembly and disassembly of signaling molecules containing SH2 domains.