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J. Biol. Chem., Vol. 276, Issue 44, 40903-40909, November 2, 2001

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Phosphorylation of the Integrin ₄ Cytoplasmic Domain Regulates Paxillin Binding^*

Jaewon Han^§¶, Shouchun Liu^§¶, David M. Rose^§, David D. Schlaepfer^**, Hayes McDonald^§, and Mark H. Ginsberg^§

From the Departments of  Vascular Biology, ^** Immunology, and ^§ Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, March 26, 2001, and in revised form, August 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

₄ integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because ₄ integrins have distinct signaling properties from other integrins. Specifically, the ₄ cytoplasmic domain binds tightly to paxillin, a signaling adaptor protein, leading to increased cell migration and an altered cytoskeletal organization that results in reduced cell spreading. The ₄ tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the ₄ tail is phosphorylated in vitro and in vivo. Furthermore, Ser⁹⁸⁸ is a major phosphorylation site. Using antibodies specific for Ser⁹⁸⁸-phosphorylated ₄, we found the stoichiometry of ₄ phosphorylation varied in different cells. However, >60% of ₄ was phosphorylated in Jurkat T cells. Phosphorylation at Ser⁹⁸⁸ blocked paxillin binding to the ₄ tail. A phosphorylation-mimicking mutant of ₄ (₄S988D) blocked paxillin binding and reversed the inhibitory effect of ₄ on cell spreading. Consequently, ₄ phosphorylation is a biochemical mechanism to modulate paxillin binding to ₄ integrins with consequent regulation of ₄ integrin-dependent cellular functions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Integrin adhesion receptors are heterodimers of  and  subunits comprised of a large extracellular domain responsible for ligand binding, a single transmembrane domain, and a cytoplasmic domain that in most cases consists of 20-70 amino acid residues (1, 2). Integrins mediate cell adhesion and participate in cell migration and cytoskeletal re-organization (1, 3). The ₄ integrins are expressed on leukocytes and their precursors, neural crest cells, and in developing skeletal muscle (4, 5). They are essential for embryogenesis, hematopoiesis, and immune responses (4-7). The ₄ integrin subunit regulates cell migration, cytoskeletal organization, and gene expression in a distinct manner from other integrin  subunits (8). ₄ integrins promote cell migration and antagonize cell spreading and contractility. These biological activities depend on the ₄ cytoplasmic domain (8). Indeed, this ₄ tail markedly stimulates cell migration and opposes cell spreading and focal adhesion formation when joined to other integrin  subunits (8).

We previously used model proteins to mimic clustered integrin cytoplasmic domains (9). Integrin tails are tethered at their N terminus to membranes spanning presumptive -helices. More importantly, they have vertical constraints, since they are initially parallel to each other and are in a specific vertical stagger as they exit the membrane. In the model proteins, clustering was mimicked by use of covalent homodimers of these domains. Helical coiled coil architecture provided the desired parallel topology and vertical stagger of the tails. Using these model proteins, we found that paxillin, a cytoplasmic signaling adaptor protein, bound tightly to the ₄ cytoplasmic domain (10). Mutations in a core nanopeptide paxillin binding sequence disrupt paxillin binding and block the ability of the ₄ tail to promote migration, oppose cell spreading, and alter cytoskeletal organization (10, 11). Furthermore, ₄₁-dependent adhesion to one of its ligands, vascular cell adhesion molecule-1 (VCAM-1), led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice (10). Consequently, the ₄-paxillin interaction plays an important role in the unusual signaling properties of ₄ integrins.

Cell migration requires rapid temporal and positional modulation of integrin-dependent cellular functions (12-14). Therefore, the importance of the ₄-paxillin interaction in cell migration suggested that it might be subject to regulation by cellular signaling events. Phosphorylation-dephosphorylation reactions are among the most widely used signaling mechanisms. Furthermore, the core paxillin binding sequence of the ₄ tail contains several potential phosphorylation sites (see Fig. 1A), leading us to assess its potential phosphorylation. In the present study, we identified phosphorylation of the ₄ tail in vitro and in vivo, mapped a major phosphorylation site to Ser⁹⁸⁸, and prepared antibodies specific for phosphorylated ₄. Stoichiometries of phosphorylation varied widely in different cell types. However, in Jurkat T cells >60% of surface ₄ was phosphorylated as assessed with the phospho-specific anti-₄ antibody. Phosphorylation at ₄ Ser⁹⁸⁸ inhibited paxillin binding to the ₄ tail and its physical association with the ₄₁ integrin. A phosphorylation-mimicking mutant of ₄ (₄S988D) blocked paxillin binding and reversed the inhibitory effect of ₄ on cell spreading. Consequently, phosphorylation of the ₄ tail at Ser⁹⁸⁸ is a novel biochemical mechanism to modify ₄ integrin-dependent cellular events.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials and DNA Constructs-- ATP and protein kinase A were purchased from Sigma. Modified trypsin (TPCK¹-trypsin) was from Promega (Madison, WI). Sulfo-NHS-biotin was from Pierce. Vectastain ABC kit was purchased from Vector Laboratories, Inc. (Burlingame, CA). ECL Western blotting detection kit was from Amersham Pharmacia Biotech. [-³²P]ATP and ³²P-inorganic phosphate were from PerkinElmer Life Sciences. The mammalian expression vector for human ₁ integrin (pHsk_1A) was a generous gift from Dr. Y. Shimizu (University of Minnesota, Minneapolis, MN). Bacterial expression vector for HA-tagged glutathione S-transferase (GST)-paxillin protein (1.7t/pGEX) was kindly provided by Drs. R. Salgia and James Griffin (Dana-Farber Cancer Center, Boston, MA). HA-tagged GST-paxillin was expressed and purified as described before (11, 15). Rabbit polyclonal antibodies specific for the cytoplasmic tail of integrin ₄ (Rb038) and for paxillin (Rb4356) were raised against the cytoplasmic tail of ₄ (KAGFFKRQYKSILQEENRRDSWSYINSKSNDD) conjugated to keyhole limpet hemocyanin and GST fusion of paxillin expressed from 1.7t/pGEX (16), respectively, and further purified by affinity chromatography using protein G-Sepharose column (Amersham Pharmacia Biotech). The following antibodies were obtained commercially: monoclonal antibody against human ₄ (HP2/1, Immunotech, Marseille, France), against paxillin (clone 349, Transduction Laboratories, Lexington, KY), against HA tag (12CA5, ATCC), and against GST (B-14, Santa Cruz Biotechnology, Santa Cruz, CA). Monoclonal antibody against human _IIb3 (D57) has been described previously (17). A prokaryotic expression vector encoding a model protein containing the wild-type ₄ integrin cytoplasmic domain has been described (10). For construction of mutant ₄, a HindIII-BamHI fragment of the ₄ integrin cytoplasmic tail was mutagenized in pBluescript vector (Stratagene, La Jolla, CA) using the QuikChange mutagenesis kit (Qiagen, Valencia, CA). The presence of the desired mutation was verified by sequencing, and the HindIII-BamHI fragment was then subcloned into the modified pET15b (9) expression vector as a HindIII-BamHI fragment. For construction of ₄ mutants in mammalian expression vector, pCDNA3.1() vector (Invitrogen, Carlsbad, CA) encoding wild-type ₄ was mutagenized using the QuikChange mutagenesis kit. Point mutations were confirmed by sequencing.

Purification of Recombinant Integrin Cytoplasmic Domains-- Production of recombinant model proteins containing the ₄ tail has been described (9). Briefly, each recombinant protein was expressed in BL21(DE3)pLysS cells (Novagen, Madison, WI), isolated by Ni²⁺-charged resins, and further purified to >90% homogeneity using a reverse-phase C18 HPLC column (Vydac, Hesperia, CA). ₄·_1A heterodimer tail mimic protein was prepared by oxidization of equimolar mixture of ₄ and _1A tail mimic proteins and isolated by reverse phase C18 HPLC. The masses of all recombinant proteins were determined by electrospray ionization mass spectroscopy (APIII, PE SCIEX, Toronto, Canada) and varied by less than 0.1% from that predicted by the desired sequence.

Cell Lines and Transfections-- Human T cell lines (Jurkat, CCRF CEM, and HuT78) and monocytic cell lines (U-937 and THP-1) were obtained from the American Type Culture Collection (ATCC). K562 cells expressing ₄₁ were generously provided by Dr. M. Hemler (Dana Farber Cancer Center, Boston, MA). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 50 units of penicillin/ml, 50 µg of streptomycin sulfate/ml, 2 mM L-glutamine, and 1% nonessential amino acids. Human lymphocytes were purified from peripheral blood from normal donors by centrifugation through a Ficoll-Paque gradient (Amersham Pharmacia Biotech) as previously described (18). Chinese hamster ovary cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% nonessential amino acid (Sigma), penicillin, and streptomycin and transiently transfected with the wild-type or mutant human ₄ constructs described above along with human ₁ construct using LipofectAMINE reagent (Invitrogen, Carlsbad, CA).

In Vitro Phosphorylation of Integrin ₄-- 1 µg of recombinant tail model proteins bound to Ni²⁺-agarose were incubated with cell lysate from Jurkat cells (50 µg of total protein) in HBB (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl₂, 0.05% Triton X-100) with protease inhibitor mixture (2 µg/ml aprotinin, 40 µg/ml bestatin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 mM Pefabloc) and phosphatase inhibitors (20 mM glycerophosphate, 50 µM sodium vanadate, 1 mM NaF, and 10 mM p-nitrophenol phosphate) for 3 h and washed several times in HBB with protease inhibitors and phosphatase inhibitors. Twenty-minute kinase reactions at 30 °C were initiated by the addition of 35 µl of kinase buffer containing [-³²P]ATP (6000 Ci/mmol) and 40 µM ATP. The bead-bound recombinant tail was then washed several times with ice-cold HBB, boiled in SDS-PAGE sample buffer, and resolved by 4-20% SDS-PAGE under reducing conditions. ³²P-labeled recombinant tail mimic proteins were visualized after autoradiography.

Metabolic Cell Labeling-- After washing with phosphate-free medium, cells were incubated for 4 h at 37 °C in phosphate-free medium containing 10% dialyzed fetal bovine serum and 0.3 mCi/ml [³²P]orthophosphate (PerkinElmer Life Sciences). Cells were washed several times with ice-cold phosphate-free medium. Cell lysates and ₄ immunoprecipitates were prepared as described below and analyzed by SDS-PAGE followed by autoradiography and Western blotting.

Immunoprecipitation and Western Blotting-- Cell lysates were routinely prepared with Nonidet P-40 lysis buffer (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 2 µg/ml aprotinin, 40 µg/ml bestatin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 mM Pefabloc, 20 mM glycerophosphate, 50 µM sodium vanadate, 1 mM NaF, 10 mM p-nitrophenol phosphate). For co-immunoprecipitation experiments, SL lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.05% Tween 20, 2 µg/ml aprotinin, 40 µg/ml bestatin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 mM Pefabloc, 20 mM glycerophosphate, 50 µM sodium vanadate, 1 mM NaF, 10 mM p-nitrophenol phosphate) was used. Lysates were held in melting ice for 30 min, and insoluble material was pelleted by centrifugation at 16,000 × g for 15 min at 4 °C. Clarified lysates were precleared with protein G-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. ₄ was then immunoprecipitated by the incubation of lysate from 2 × 10⁷ cells with 1 µg of anti-₄ monoclonal antibody (HP2/1, Immunotech) for 2 h at 4 °C followed by incubation with goat anti-mouse IgG-Sepharose for at least 2 h at 4 °C. Immunoprecipitates were washed several times in the same lysis buffer, boiled in 1× SDS-PAGE sample buffer, then separated by 4-20% SDS-PAGE (Invitrogen, Carlsbad, CA). Proteins were electrophoretically transferred to either nitrocellulose membrane (Bio-Rad) (100 V, 1 h 30 min) or polyvinylidene fluoride membrane (Millipore, Bedford, MA). Membranes were washed twice in Tris-buffered saline with 0.1% Tween 20 (TBST), blocked with 5% nonfat dry milk in TBST, and incubated with antibodies for 2 h. Antibody binding was detected using horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies and visualized with ECL chemiluminescence reagents (Amersham Pharmacia Biotech). For re-probing with other antibodies, blots were stripped with stripping buffer (62.5 mM Tris-HCl, pH 6.8, 100 µM -mercaptoethanol, 2% SDS) at 65 °C for 30 min. After washing several times in TBST, the membranes were blocked with 5% nonfat dry milk containing TBST and reprobed as described above. To label surface molecules, cells were washed three times with phosphate-buffered saline and then incubated with 0.5 mg/2 × 10⁷ cells/ml of sulfo-NHS-biotin (Pierce) for 30 min at room temperature. Unreacted biotin was quenched and washed from the cells with TBS (0.1 M Tris-HCl, pH 7.4, 150 mM NaCl). Biotinylated proteins were separated by SDS-PAGE gel, transferred to nitrocellulose membranes, and detected using Vectastain ABC kit and ECL chemiluminescence.

Phosphoaminoacid Analysis and Phospho-peptide Mapping-- Phosphoproteins extracted from SDS-PAGE gels were hydrolyzed in 6 N HCl for 1 h at 110 °C. After lyophilization, phosphoamino acids were separated by two-dimensional thin layer electrophoresis (HTLE 7000, CBS Scientific) along with a phosphoamino acid standards mixture on glass-backed cellulose thin layer chromatography plates (EM Science) using 0.58 M formic acid and 1.36 M glacial acetic acid, pH 1.9, in the first dimension and 0.87 M glacial acetic acid, 0.5%(v/v) pyridine, and 0.5 M EDTA, pH 3.5, in the second dimension. After visualization of the markers by ninhydrin spraying, the plate was exposed to film (19). Phospho-peptide mapping was performed as described by Boyle et al. (19) and Luo et al. (20). Briefly, labeled ₄ was immunoprecipitated from ³²P-labeled cells and separated in SDS-PAGE as described above and electro-transferred to nitrocellulose membrane. The membrane was then exposed to film to localize ₄. The band corresponding to ₄ was then cut out and incubated with 0.5% polyvinylpyrrolidone 360 (PVP360, Sigma) dissolved in 0.1 M acetic acid for 30 min at 37 °C. Membrane fragments were washed five times with water and finally with freshly made 50 mM NH₄HCO₃, pH 8.0, and treated with 10 µg of TPCK-trypsin (in 0.1 mM HCl) in 200 µl of 50 mM NH₄HCO₃ overnight at 37 °C. For complete digestion, an additional 10 µg of TPCK-trypsin was added, and the tube was incubated for another 3 h. After the incubation, the remaining NH₄HCO₃ was removed by repeated lyophilization in a SpeedVac, and the peptides were oxidized by incubation in performic acid. Phospho-peptides dissolved in a small volume of distilled water were spotted on a glass-backed cellulose plate and then separated in the first dimension by electrophoresis, pH 1.9, and separated in the second dimension by ascending chromatography in phospho-chromatography buffer (21). The phospho-peptides were visualized by exposing plates to Kodak XAR MS film for ~3 days at 80 °C with intensifying screens.

Generation of Phospho-specific Anti-₄ Antibody-- The peptide RDS⁹⁸⁸WSYINSK was synthesized with or without a phosphorylation at Ser⁹⁸⁸. Both peptides were purified by reversed-phase HPLC, and their identities were confirmed by mass spectrometry. The synthetic peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as the coupling reagent. Rabbits were immunized by intracutaneous injection of the conjugate and were bled at bi-weekly intervals. Antibodies were rendered phospho-specific by absorption with the ₄ tail model protein immobilized on Ni²⁺ chelate resin. Specificities of the final antibodies were verified by immunoblotting.

Measuring the Stoichiometry of Ser Phosphorylation-- Jurkat cells were surface-biotinylated by sulfo-NHS-biotin as described above, and phosphorylated ₄ was quantitatively precipitated from the Jurkat cell lysate in a preclearing step either with PS₄ antibody or with rabbit IgG. Afterward, the remaining ₄ in the cell lysate was immunoprecipitated with HP2/1. Immunoprecipitated ₄ was separated in a SDS-PAGE gel (4-20%) and transferred to nitrocellulose membranes and visualized by chemiluminescence after staining with Vectastain ABC. Densitometry (AlphaImager 2000, Alpha Innotech Corp.) was used for quantitative comparisons.

Cell Spreading Assay-- Cell spreading was performed as described (10, 11). Briefly, Chinese hamster ovary cells were transiently transfected with wild type or S988D mutant human integrin ₄ in combination with human ₁. Equal expression of mutant and wild-type ₄ integrins was observed by fluorescence-activated cell sorter analysis. Cells resuspended in Dulbecco's modified Eagle's medium with 1% bovine serum albumin were plated on coverslips coated with 5 µg/ml recombinant CS-1, an ₄ integrin binding fragment of fibronectin, in 12-well plates and, after spreading for 2 h at 37 °C, were assessed by phase contrast microscopy. Cells that exhibited flattening and the presence of lamellipodia were scored as spreading cells. Digital images were acquired with an Olympus IX70 microscope equipped with CoolSnap digital color camera.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Phosphorylation of the Integrin ₄ Subunit Cytoplasmic Domain-- The ₄ integrin cytoplasmic domain contains multiple potential phosphorylation sites. To investigate possible ₄ phosphorylation, we first assessed the capacity of cell extracts to phosphorylate the ₄ tail in vitro. A Jurkat T cell extract phosphorylated the ₄ cytoplasmic domain but not those of integrins _IIb or _1A (Fig. 1B). Because integrins are heterodimers, the ₄ tail is paired with that of the  subunit (for example, integrin _1A). However, the ₄ tail was phosphorylated by Jurkat T cell lysate when in a heterodimer formed with the _1A tail. Thus, the ₄ tail is phosphorylated in vitro by a T cell extract in the presence or absence of the _1A tail.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Integrin 4 is phosphorylated on its cytoplasmic tail in vitro and in vivo. A, schematic representation of the 4 tail (residue 968-999). All the possible putative phosphorylation sites are indicated as bold and underlined characters. Previously mapped paxillin binding motif (11) is boxed. B, recombinant tail mimic proteins of 4 or 41 along with IIb immobilized on Ni2+-agarose beads were phosphorylated in vitro by lysate from Jurkat cells as described under "Experimental Procedures" and subjected to SDS-PAGE. Phosphorylated proteins were detected by autoradiography. C, phosphorylation of 4 in various cells. Immunoprecipitated (IP) 4 proteins from the 32P-labeled cells were subjected to SDS-PAGE and visualized by autoradiography. 4 was identified by Western blotting using anti-4 antibody (Rb038). Note that two 4 bands appeared in the 4 blot; however, the intensity of these two bands did not correlate with the extent of 4 phosphorylation. The lower band is probably an 4 precursor, since it is not observed in immunoprecipitates formed with anti-4 antibodies from surface-labeled cells (data not shown).

To assess in vivo phosphorylation of ₄, we metabolically labeled Jurkat T cells with [³²P]orthophosphate. Immunoprecipitated ₄ was biosynthetically phosphorylated in these cells (Fig. 1C). We examined several T cell lines and observed constitutive ₄ phosphorylation in Jurkat and CEM (Fig. 1C). ₄ was also phosphorylated in all of the monocytic cells that we examined (U937 and THP-1) and in peripheral blood T cells as well (data not shown). Furthermore, treatment of Jurkat cells with agonists such as anti-CD3+ anti-CD28 or phorbol myristate acetate did not increase ₄ phosphorylation (data not shown). However, several cell lines exhibited little or no ₄ phosphorylation. These included HuT78, rat basophilic leukemia, and ₄-transfected K562 cells. Nevertheless, the ₄ cytoplasmic domain was present in ₄ immunoprecipitates formed from these cells (Fig. 1C). Thus, the ₄ tail is constitutively phosphorylated in vivo in some cells but not in others.

Ser⁹⁸⁸ Is a Major ₄ Phosphorylation Site-- To map the phosphorylation site(s) of ₄, we first carried out phosphoamino acid analysis of metabolically labeled ₄ immunoprecipitated from Jurkat T cells (Fig. 2A, left panel) and of recombinant ₄ tail labeled by an in vitro kinase reaction (Fig. 2A, right panel). In both preparations, only phosophoserine was detected (Fig. 2A). Thus, the ₄ tail is phosphorylated on serine residues in vitro and in vivo.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Integrin 4 is phosphorylated on Ser988. A, phosphorylated 4 was subjected to phosphoamino acid analysis using two-dimensional electrophoresis as described under "Experimental Procedures." 4 proteins, immunoprecipitated from 32P-labeled Jurkat cells (left panel) or recombinant 4 tail mimic proteins phosphorylated in vitro in the presence of [-32P]ATP were separated by SDS-PAGE and transferred to the membrane. After exposure to film, the band corresponding to 4 was excised and hydrolyzed in 6 N hydrochloric acid for 1 h at 110 °C. Phosphoamino acids were separated by two-dimensional electrophoresis on a cellulose plate at pH 1.9 in the first dimension and at pH 3.5 in the second dimension. PS, PT, and PY indicate phosphoserine, phosphothreonine, and phosphotyrosine, respectively. B, recombinant model proteins immobilized on Ni2+-agarose beads were phosphorylated in vitro by recombinant PKA and subjected to SDS-PAGE. Phosphorylated proteins were detected by autoradiography. C, integrin 4, in vitro phosphorylated by PKA (left panel) or immunoprecipitated 4 from 32P-labeled Jurkat cells (right panel), were subjected to phospho-peptide mapping. Phosphorylated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and visualized by autoradiography. 4 bands were excised and digested with trypsin, followed by two-dimensional mapping using electrophoresis, pH 1.9, in the first dimension and ascending chromatography in the second dimension as described under "Experimental Procedures." D, recombinant tail mimic proteins of wild type and 4/S988A along with IIb immobilized on Ni2+-agarose beads were phosphorylated by either recombinant PKA or Jurkat cell lysate in vitro and subjected to SDS-PAGE. Phosphorylated 4 was visualized by autoradiography as described above.

The ₄ tail contains 5 serine and 2 tyrosine potential phosphorylation sites (Fig. 1A). A search of the data base of consensus sequence motifs for the potential phosphorylation sites in the ₄ tail using PBASE (www.cbs.dtu.dk/htbin/pbasepredict.pl) identified an exact recognition site for protein kinase A (PKA) (XRRXS, where X indicates any amino acid,  indicates a hydrophobic residue, and underlining indicates the site of phosphorylation) around Ser⁹⁸⁸ (RRDSWS) (22). To learn whether ₄ was a PKA substrate, we assessed the capacity of PKA to phosphorylate the ₄ tail in vitro. Both ₄ and the ₄₁ heterodimer cytoplasmic domains were phosphorylated by purified recombinant PKA (Fig. 2B).

To determine whether Ser⁹⁸⁸ is phosphorylated in vivo, ₄ was metabolically labeled by ³²P in Jurkat T cells. The ₄ subunit was isolated by immunoprecipitation followed by SDS-PAGE. Tryptic digests of the isolated subunit were then subjected to two-dimensional phospho-peptide mapping. The phospho-peptide patterns were compared, with those observed with the ₄ tail phosphorylated by PKA in vitro. A major co-migrating phospho-peptide spot, labeled 1 in Fig. 2C, was observed (PKA phosphorylated ₄ in the left panel and metabolically labeled ₄ in the right panel). Thus, the major tryptic ₄ phospho-peptide isolated from Jurkat T cells is also present in PKA-phosphorylated ₄. As noted above, ₄ Ser⁹⁸⁸ is a predicted PKA phosphorylation site, and the major ₄ phospho-peptide from Jurkat cells co-migrates with one from PKA-phosphorylated ₄. These data suggest that ₄ Ser⁹⁸⁸ is a major phosphorylation site in vivo. To test the importance of Ser⁹⁸⁸ in phosphorylation, we assessed in vitro phosphorylation of an ₄/S988A mutant by both PKA and a Jurkat cell lysate (Fig. 2D). This substitution completely abolished ₄ phosphorylation by PKA and eliminated ~90% of the ₄ phosphorylation by the Jurkat cell lysate. Taken together, these results indicate that Ser⁹⁸⁸ is a major phosphorylation site in the ₄ tail.

Regulation of Paxillin Binding by Phosphorylation of ₄ Ser⁹⁸⁸-- Ser⁹⁸⁸ resides in the middle of the paxillin binding sequence of the ₄ tail (Fig. 1A). Consequently, we asked whether phosphorylation of Ser⁹⁸⁸ could regulate paxillin binding. We examined the binding of recombinant paxillin to ₄ tail model proteins that had been phosphorylated in vitro with PKA to stoichiometries of 0.95 mol of PO/mol of ₄ tail. Phosphorylation of ₄ abolished paxillin binding. Furthermore, paxillin binding could be reconstituted by dephosphorylation with alkaline phosphatase (Fig. 3A). Thus, PKA phosphorylation in vitro blocks the binding of the ₄ tail to paxillin.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of 4 cytoplasmic tail on Ser988 inhibits paxillin binding to 4. A, recombinant 4 tail mimic protein bound to Ni2+-agarose bead was phosphorylated by PKA in vitro as described above. Some of the phosphorylated 4 protein was then subjected to in vitro phosphatase reaction using alkaline phosphatase (AP) for 30 min at 30 °C in phosphatase buffer (50 mM Tris HCl, pH 7.4, 1 mM MgCl2). Phosphorylated or dephosphorylated 4 tail model protein was incubated with GST-paxillin in PN lysis buffer (10 mM PIPES, pH 6.8, 50 mM NaCl, 150 mM sucrose, 50 mM NaF, 40 mM sodium pyrophosphate, 1 mM sodium vanadate, 1% Triton X-100, and protease inhibitor mixture). After a 30-min incubation at room temperature, unbound protein was washed, and pellets were subjected to SDS-PAGE. GST-paxillin was detected by Western blotting using anti-HA antibody (GST-paxillin also has an HA tag). B, 32P-labeled Jurkat cells were surface-biotinylated and lysed with SL lysis buffer (described under "Experimental Procedure") in the presence or absence of phosphatase inhibitors (20 mM glycerophosphate, 1 mM NaF, 10 mM p-nitrophenol phosphate, 50 µM sodium vanadate). Then paxillin was immunoprecipitated (IP), and coimmunoprecipitated 4 was visualized by blotting for biotin after separation in SDS-PAGE gel (upper panel). Immunoprecipitated paxillin was detected by anti-paxillin antibody. C, 4 was immunoprecipitated from the same Jurkat cell lysate used in panel B and resolved in an SDS-PAGE gel. Phosphorylated 4 was detected by autoradiography, and its identity was verified by Western blotting for 4 (Rb038) (lower panel).

As noted above, ₄ Ser⁹⁸⁸, the PKA phosphorylation site, is a major target of phosphorylation in Jurkat T cells. To determine whether phosphorylation of intact ₄ regulated paxillin binding, we assessed the co-precipitation of ₄ with paxillin. In previous studies (10), we found that nearly 100% ₄₁ in Jurkat T cells could associate with paxillin when the cell lysates were prepared in the absence of phosphatase inhibitors. However, inclusion of phosphatase inhibitors resulted in a 2-3-fold decrease in ₄ co-precipitated with paxillin (Fig. 3B). This decrease correlated with a 2-3-fold increase in ₄ phosphorylation under these conditions (Fig. 3C). Thus paxillin binding to the ₄ tail is regulated by phosphorylation of Ser⁹⁸⁸.

₄ Ser⁹⁸⁸ Can Be Phosphorylated to High Stoichiometries in Vivo-- The previous results suggested that ₄ phosphorylation at Ser⁹⁸⁸ could regulate paxillin binding. To evaluate the potential biological relevance of this observation, we assessed the stoichiometry of ₄ phosphorylation. To quantitatively assay ₄ phosphorylation, we raised a polyclonal antibody against an ₄ synthetic peptide containing phosphoserine at Ser⁹⁸⁸. This antibody was rendered phospho-specific by absorption with nonphosphorylated ₄ tail. The absorbed antibody (PS₄) failed to react with ₄ tail but did so when the ₄ tail was phosphorylated with PKA (Fig. 4A). To further assess the specificity of this antibody for phospho-₄, we also examined whether PS₄ recognizes a phosphorylated serine residue in the context of a similar flanking sequence. As shown in Fig. 4A (right panel), this antibody did not cross-react with a GST fusion protein phosphorylated in its PKA recognition site (RRASV). Furthermore, PS₄ reacted with phospho-₄ isolated from Jurkat T-cells (Fig. 4B). Reactivity was abolished by dephosphorylation of the isolated ₄ with alkaline phosphatase (Fig. 4B). Thus, the PS₄ specifically reacts with phosphorylated but not with non-phosphorylated ₄ in a sequence-specific manner.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Production of phospho-specific anti-4 antibody for assessment of the stoichiometry of 4 phosphorylation. A, specificity of Ser988 phosphorylation-specific antibody by Western blotting. Recombinant 4 model proteins immobilized on Ni2+-agarose beads were phosphorylated by PKA in vitro for the indicated incubation times in the presence of [-32P]ATP. After washing, bead-bound proteins were resolved in SDS-PAGE, and phosphorylation of 4 was detected either by Western blotting using phospho-specific rabbit polyclonal anti-4 antibody (4*) or by autoradiography. In a similar experiment (left panel), a GST fusion protein, which contains a recognition site for PKA, was phosphorylated, and its phosphorylation was visualized by autoradiography and Western blotting with phospho-specific rabbit anti-4 antibody. B, specificity of Ser988 phosphorylation-specific antibody by Western blotting of immunoprecipitated (IP) 4. 4 was immunoprecipitated from 32P-labeled Jurkat cells. After washing pellets, immunoprecipitates were resuspended in phosphatase buffer and incubated for the indicated times with alkaline phosphatase (AP) at 30 °C. 4 immunoprecipitates were subjected to SDS-PAGE, and phosphorylation of 4 was visualized by Western blotting with phospho-specific anti-4 antibody (4*) or by autoradiography. The presence of 4 in the immunoprecipitates was assessed by Western blotting with an anti-4 antibody (Rb038) directed against the 4 tail. C, estimation of stoichiometry of 4 phosphorylation. Lysates of surface-biotinylated Jurkat cells were prepared in the presence or absence of phosphatase inhibitors. The lysates were precleared by immunoprecipitation with either phospho-specific anti-4 antibody (PS4) or irrelevant rabbit IgG, and the remaining 4 was immunoprecipitated by an antibody reactive with the extracellular domain of 4(HP2/1). Immunoprecipitates were resolved in SDS-PAGE, and biotinylated 4 was detected by transfer to nitrocellulose membranes and staining with Vectastain ABC (upper panel). Depicted is the result of one of two experiments with similar results. Densitometry was used for quantitative comparisons (lower panel). 4* indicates phosphorylated 4.

We used the PS₄ antibody to assess the stoichiometry of ₄ phosphorylation. Preclearing cell lysates with PS₄ removed 60% of the total ₄ from Jurkat T cells (Fig. 4C). In sharp contrast, when ₄ was dephosphorylated by omission of phosphatase inhibitors, preclearing with PS₄ antibody did not deplete ₄ (Fig. 4C). Phosphorylation of ~60% of ₄ in Jurkat cells can account for the 2-3-fold reduction in paxillin-associated ₄ when phosphatase inhibitors are present (see Fig. 3B). Consequently, at least 60% of ₄ is constitutively phosphorylated in these Jurkat T cells, and phosphorylated ₄ manifests reduced association with paxillin.

A Mutation That Mimics ₄ Phosphorylation Disrupts Paxillin Binding and Promotes Cell Spreading-- To further address the functional role of ₄ phosphorylation at Ser⁹⁸⁸, we generated an ₄ mutant that mimics constitutively phosphorylated status (S988D). To confirm that the aspartic acid substitution mimicked the biochemical effects of ₄ phosphorylation, we examined the binding of recombinant paxillin to wild-type and mutant ₄ tail model proteins. Aspartic acid substitution at Ser⁹⁸⁸ markedly reduced paxillin binding (Fig. 5A). To assess a biological consequence of ₄ phosphorylation, we examined the effect of this mutation on ₄-dependent cell spreading. Paxillin binding to ₄ cytoplasmic domain inhibits cell spreading, and therefore, disruption of this interaction results in increased cell spreading (10, 11). To examine the effect of the S988D mutation on cell spreading, we transiently transfected Chinese hamster ovary cells with either ₄ or ₄(S988D). Cells transfected with ₄(S988D) spread promptly on the CS-1 fragment of fibronectin, a ligand of integrin ₄₁ (Fig. 5, B and C). In sharp contrast, cell spreading was markedly retarded in cells expressing the wild type ₄ (Fig. 5, B and C). Thus, phosphorylation of ₄S⁹⁸⁸ leads to loss of paxillin binding with consequent alterations in cellular response to ₄₁-mediated adhesion.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   S988D mutation in 4 cytoplasmic domain disrupts paxillin binding to 4 and increases 4-dependent cell spreading. A, effect of S988D mutation on paxillin binding to 4 cytoplasmic domain. Immobilized wild-type (wt) or mutant 4 tail or IIb tail model protein was incubated with GST-paxillin in PN lysis buffer (10 mM PIPES, pH 6.8, 50 mM NaCl, 150 mM sucrose, 1% Triton X-100, and protease inhibitor mixture) for 30 min at room temperature. The Ni2+ bead-bound proteins were fractionated by SDS-PAGE, and bound paxillin was detected by Western blotting. Bound paxillin was quantified by densitometry, and background binding to the IIb tail was subtracted in each experiment. The data represent the mean ± S.D. of three separate experiments. B, effect of S988D mutation on cell spreading. Wild type and mutant Chinese hamster ovary transfectants were allowed to spread on the CS-1 fragment of fibronectin for 2 h. Images of typical fields were acquired at a magnification of 400× (upper panels). Spread cells were enumerated as described under "Experimental Procedures." The data represent the mean ± S.D. of triplicate determinations (lower panel).

In this study, we found that integrin ₄ is phosphorylated on a serine residue(s) and that Ser⁹⁸⁸ is a major target of phosphorylation. Phospho-peptide mapping showed that the major ₄ phospho-peptide was that containing Ser⁹⁸⁸. Furthermore, the S988A mutation abolished ~90% of ₄ phosphorylation by Jurkat cell lysate. The tryptic phospho-peptide contains two other potential phosphorylation sites (Ser⁹⁹⁰ and Ser⁹⁹⁴). However, immunoprecipitation and immunoblotting with PS₄ antibody verified the phosphorylation on Ser⁹⁸⁸. Consequently, Ser⁹⁸⁸ is a major site of phosphorylation. Our data show that this site is used by PKA in vitro. However, we emphasize that this site could be a target of other kinases (e.g. Ca²⁺/calmodulin-dependent protein kinase II, protein kinase G) in vivo. The development of the Ser⁹⁸⁸ phospho-specific antibody should facilitate the identification of the relevant kinases.

Our studies identify ₄ phosphorylation at Ser⁹⁸⁸ as a potential regulator of ₄ integrin signaling. In particular, the interaction of ₄ with paxillin (or one of its paralogs) is required for ₄ specific promotion of cell migration, enhanced phosphorylation of pp125^FAK (focal adhesion kinase (FAK)) and reduction of cell spreading (10). Phosphorylation at Ser⁹⁸⁸ abolishes this interaction. Consequently, the phosphorylation will modulate all of the responses dependent on the ₄-paxillin interaction. Previous studies report regulatory phosphorylation of integrin tails. For example, Tyr phosphorylation of ₃ and _1A tails regulates integrin signaling and migration (23, 24). The effects of ₃ Tyr phosophorylation may be because of altered interactions with either myosin or the SHC adaptor (25, 26). In addition, ₃ phosphorylation at Thr⁷⁵³ has been proposed to regulate bi-directional integrin signaling (27, 28). _L and _M can be phosphorylated on their serine residues (29-31), and these phosphorylations may regulate cytoskeletal associations. Furthermore, ₃ and ₆ Ser phosphorylation may regulate cell shape and cell migration (32, 33). However, the effects of these  subunit phosphorylations on specific biochemical interactions of the  tails have not been defined. In the present study, we have identified a high stoichiometry phosphorylation of an integrin  tail that regulates a protein-protein interaction that controls biological responses. Temporal and spatial regulation of ₄ phosphorylation may thus be an important biochemical mechanism for the control of ₄-mediated cell functions.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health (to M. H. G.), Juvenile Diabetes Foundation International (to D. M. R.), and the Arthritis Foundation (to J. H.), an American Heart Association Scientist Development Grant (to S. L.), NCRR Grant RR11823-5 and a Merck Genome Research Institute grant (to H. M.), and National Institutes of Health Grant CA-75240 (NCI) (to D. D. S.). This is publication number 14054-VB from the Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

Current address: COR Therapeutics, Inc., South San Francisco, CA 94080.

To whom correspondence should be addressed: Dept. of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7124; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.

Published, JBC Papers in Press, August 30, 2001, DOI 10.1074/jbc.M102665200

	ABBREVIATIONS

The abbreviations used are: TPCK, L-1-tosylamide-2-phenylethylchloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBST, TBS with Tween; PKA, protein kinase A; HA, hemagglutinin; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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