From the Division of Immunology and ¶ Howard
Hughes Medical Institute, Children's Hospital, Harvard Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
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Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. In this report, we demonstrate that recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASP), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASP-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. We demonstrate the presence of profilin in Nck precipitates suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin.
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INTRODUCTION |
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Nck is a 47-kDa protein ubiquitously expressed in mammalian cells (1) and is composed of three tandem Src homology 3 (SH3)1 domains followed by a single SH2 domain. Nck has no intrinsic catalytic activity and acts as an adaptor molecule to couple upstream signals, usually those initiated by activation of receptor tyrosine kinases (RTKs), to downstream signal transducer molecules.
Ligand binding to RTK induces the receptor chains to dimerize and to transphosphorylate on specific tyrosine residues that provide docking sites for SH2 domains (2). Nck interacts via its SH2 domain with phosphotyrosine residues in RTKs such as the receptors for epidermal growth factor, platelet-derived growth factor, vascular endothelial cell growth factor, and ephrin receptors (EphB1 and EphB2) (3-7) or in protein substrates of RTKs such as insulin receptor substrate-1 (8). Nck interacts via its SH3 domains with effector molecules containing proline-rich sequences bringing them to the proximity of ligandactivated RTKs.
The three SH3 domains (SH3.1, SH3.2, and SH3.3) of Nck interact
selectively with target proteins. The SH3.1 domain mediates Nck
association with the Nck-associated protein 1 (Nap1) (9). The SH3.2
domain mediates Nck interaction with p21-associated kinase (10, 11),
Sos, a guanine nucleotide exchange factor for Ras (12), the
serine/threonine kinase PRK2/NAK (13, 14), and Nck, Ash-, and
phospholipase C-binding protein 4 (15). The SH3.3 domain mediates
Nck interaction with the Wiskott-Aldrich syndrome protein (WASP) (16),
and the
isoform of the serine/threonine kinase casein kinase I
(CKI-
2) (17). Other proteins that have been shown to interact with
Nck but for which the specific SH3 domain that mediates binding has not
been defined include c-Cbl (18), focal adhesion kinase (19), pp105, a
lymphocyte-type CRK-associated substrate that binds to FAK and Crk
(20), and Nck interacting kinase (21). Recently a nuclear protein, SAM 68, has been identified as a specific binding partner of nuclear Nck
(22). The SH3 domains of Dock, the Drosophila homologue of
Nck, have been shown to interact with the Drosophila
protein-tyrosine-phosphatase dPTP61F (23).
Cytoskeletal rearrangement is triggered by a variety of external stimuli such as growth factors, stress, and adhesion through integrins (24) and is mediated by small GTPases. In mammalian cells, Rho family GTPases control the reorganization of the actin cytoskeleton in response to growth factors. For example, epidermal growth factor and platelet-derived growth factor activate Rac which induces ruffling of the cell membrane with lamellipodia formation (24). Polymerization of actin filaments in the cytosol is orchestrated by secondary messengers of signal transduction pathways and by proteins that interact with actin. Profilin is a 15-kDa G-actin-binding protein that regulates actin filament assembly. Profilin promotes actin polymerization by favoring the exchange of ADP to ATP on actin (25) and by lowering the critical concentration of ATP-actin (26). Profilin can also contribute to the pool of unassembled actin when barbed ends are capped (27).
Recently, we have identified a novel WASP-interacting protein (WIP) (28). WIP is a widely expressed 503-amino acid long protein with homology in its amino-terminal sequence to the yeast protein verprolin which is involved in cytoskeleton organization (29). WIP overexpression increases the basal level of polymerized actin in human lymphoid cells and induces the formation of actin-rich cerebriform projections on the cell surface (28). WIP is proline-rich and contains potential SH3 domain-binding sequences. In an effort to investigate the role of WIP in signal transduction, we analyzed WIP binding to Nck. Our results show that Nck binds to WIP and suggest that WIP-Nck interaction may bridge cell-surface receptors to the actin cytoskeleton.
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EXPERIMENTAL PROCEDURES |
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Yeast Two-hybrid System-- Full-length Nck cDNA was cloned in-frame into the bait vector pGBT9 (CLONTECH). The sequence of the clone was confirmed by DNA sequence analysis, and the clone was designated Nck-GBT9.
WIP4 is a truncation of WIP cDNA that encodes the carboxyl-terminal portion of WIP (amino acids 321-503) (28). WIP4 cDNA cloned in the yeast two-hybrid vector pGAD was used to construct the WIP4 deletion mutants. Deletions were obtained by digestion with the appropriate restriction enzymes followed by Klenow treatment and religation. pGAD-WIP-(321-415) was obtained by digestion with StuI and PstI, and pGAD-WIP-(321-376) was obtained by digestion with SfiI and PstI, and pGAD-WIP-(377-503) was obtained by SfiI digestion, Klenow treatment, EcoRI digestion, and a second Klenow treatment. WIP inserts for pGAD-WIP-(416-503) and pGAD-WIP-(416-488) constructs were obtained by polymerase chain reaction. All constructs were confirmed by sequencing. Yeast transformation and colony analysis were performed according to the manufacturer's instructions (Matchmaker Two-Hybrid System Protocol, CLONTECH).GST Fusion Proteins--
Glutathione S-transferase
(GST) fusion proteins of Nck and of each of its three SH3 domains were
generated as described previously (11). All expression constructs were
verified by DNA sequence analysis. Expression of fusion proteins in
transformed Escherichia coli was induced for 2 h with
0.1 mM
isopropyl-thio-D-galactopyranoside. Fusion proteins
were purified as described previously (28).
Generation of WIP Expressing BJAB Cells-- WIP4 cDNA was cloned into a modified pcDNA3 vector that expresses cloned cDNA as an amino-terminal FLAG fusion protein and was transfected into the human B lymphoma cell line BJAB as described (28). The culture medium for BJAB-transfected cells was supplemented with 1.5 mg/ml G418 (Calbiochem).
Affinity Precipitation of WIP by GST Fusion Proteins-- Lysates of BJAB cells transfected with pcDNA3 or with pcDNA-WIP4 were obtained as described previously (28) and precleared for 1 h with 25 µl of GST-Sepharose (Amersham Pharmacia Biotech). Supernatants were tumbled for 16 h with 2 µg of GST or GST fusion proteins immobilized on GSH beads. The beads were washed, suspended in Laemmli loading buffer and subjected to PAGE on 4-15% gradient gels and Western blotting. The blots were developed with rabbit anti-WIP followed by protein A conjugated to horseradish peroxidase or with anti-FLAG M2 mAb followed by goat anti-mouse conjugated to horseradish peroxidase and enhanced chemiluminescent detection (ECL).
Immunoprecipitation of FLAG-WIP from BJAB Cells-- BJAB cells or BJAB cells transfected with pcDNA-WIP were washed twice with phosphate-buffered saline and lysed (45 × 106 cells in 0.35 ml) in ice-cold lysis buffer (50 mM Tris, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 30% glycerol, 0.4 mM Na3VO4, 10 mM NaF, 10 mM Na3P2O7, protease inhibitor mixture (Complete, Boehringer Mannheim) and 1% Brij 96) for 30 min. Lysates were centrifuged at 16,000 × g for 15 min at 4 °C and precleared for 1 h at 4 °C with 5 µl of normal mouse serum bound to protein G-Sepharose (Amersham Pharmacia Biotech) and then incubated overnight at 4 °C with 8 µg of anti-FLAG M2 monoclonal antibody (mAb) or of isotype-matched control MOPC21 mAb preadsorbed onto 40 µl of protein G-Sepharose. The precipitates were washed 4× with modified lysis buffer containing 10% glycerol and 0.2% Brij-96, eluted in Laemmli loading buffer, and subjected to SDS-PAGE on 4-15% gradient gels and Western blot analysis with anti-FLAG mAb or anti-Nck mAb (Transduction Laboratories). The blots were developed by ECL as described above.
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RESULTS AND DISCUSSION |
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Nck Interacts with WIP in the Yeast Two-hybrid System-- WIP contains several proline-rich sequences including three repeats of the sequence GRSGPXPPXP. This sequence is repeated twice in WASP and is involved in the binding of WASP to the SH3.3 domain of Nck (30). We therefore reasoned that WIP may be a candidate for binding to Nck. Since all the three GRSGPXPPXP sequences were present within WIP4, a truncation of WIP that contains amino acids 321-503, we tested the interaction of WIP4 with Nck by the yeast two-hybrid system. Table I shows that Nck interacts specifically with WIP4. Nck did not interact with human TRAF1 (tumor necrosis factor receptor-associated factor 1) used as a control, and WIP4 did not interact with laminin (Table I). As expected, WIP4 interacted with WASP (28).
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Endogenous Nck Co-immunoprecipitates with WIP from BJAB Cells-- To demonstrate the Nck-WIP association in vivo, we examined whether Nck and WIP co-immunoprecipitate from cells. To this purpose, we examined the presence of Nck in anti-FLAG immunoprecipitates of lysates from human B cells BJAB transfected with FLAG-tagged WIP4 cloned in pcDNA3. Fig. 1 shows the presence of Nck in anti-FLAG immunoprecipitates from FLAG-WIP4 transfected cells (lane 1). Nck was not detected in MOPC21 mAb immunoprecipitates of WIP4-transfected cells (lane 2) nor in M2 immunoprecipitates of untransfected BJAB cells (lane 3). To ascertain the presence of FLAG-tagged WIP in the immunoprecipitates, the membrane was stripped and reblotted with anti-FLAG M2 mAb (Fig. 1, lower panel). FLAG-tagged WIP4 is detected in M2 immunoprecipitates from BJAB cells transfected with FLAG-WIP4 (lane 1) and, as expected, in total lysates from FLAG-WIP4-transfected cells (lane 4). FLAG-WIP4 was neither detected in MOPC21 immunoprecipitates from WIP4-transfected cells (lane 2) nor in M2 immunoprecipitates from untransfected cells (lane 3). Treatment of cells with phorbol 12-myristate 13-acetate for 15, 30, or 60 min did not alter the capacity of Nck and WIP to co-immunoprecipitate (data not shown) suggesting that Nck phosphorylation induced by phorbol 12-myristate 13-acetate (31) does not regulate WIP-Nck interaction.
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WIP Binds to the Second SH3 Domain of Nck-- To confirm Nck interaction with full-length WIP, we used GST-Nck fusion protein to affinity precipitate endogenous WIP from BJAB cells. The precipitates were run on SDS-PAGE and Western-blotted with anti-WIP rabbit antibody. Fig. 2A shows that WIP is present in GST-Nck precipitates but not in control GST precipitates.
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Mapping of the Nck-binding Site of WIP-- We have previously shown that WASP binds to the carboxyl-terminal region of WIP, amino acids 377-503 (28). To determine whether the WASP- and Nck-binding sites on WIP overlap, we examined the interaction of WIP deletion mutants with WASP and Nck using the yeast two-hybrid system. Fig. 3 shows that WIP-(416-488) binds to WASP but not to Nck. In contrast, the WIP deletion mutant WIP-(321-415) binds to Nck but not to WASP. Taken together, these results show that the WASP and Nck binding domains of WIP differ.
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WIP May Bridge Nck to Profilin and the Cytoskeleton-- WIP interacts with profilin (28). The two profilin binding consensus sequences in WIP (APPPPP) are located at positions 8-13 and 427-432 and are outside the Nck-binding site (amino acids 321-415). This raised the possibility that WIP may couple Nck to profilin. We therefore examined whether profilin co-precipitates with Nck. Fig. 4 shows that endogenous profilin from lysates of BJAB cells is bound to GST-Nck but not to GST. Nck lacks proline-rich sequences, including profilin binding consensus sequences (A, G, L, or S followed by PPPPP) (32) and fails to interact with profilin in the yeast two-hybrid system (data not shown). These results suggest that the binding of profilin to Nck is indirect and may be mediated by WIP, although we cannot rule out a role for other intermediaries.
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ACKNOWLEDGEMENT |
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We thank Dr. Erdyni Tsitsikov for the TRAF1-pGAD424 construct.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL-59561-01 and AI-37130 (to N. R.) and by grants from the Baxter, Olsten, and Alpha Therapeutics Corp.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.
§ Supported by a fellowship from Fundación Ramón Areces, Madrid, Spain.
To whom correspondence should be addressed: Enders 8, Division
of Immunology, 300 Longwood Ave., Children's Hospital, Boston, MA
02115. Tel.: 617-355-7602; Fax: 617-355-8205; E-mail:
geha{at}a1.tch.harvard.edu.
The abbreviations used are: SH3, Src homology 3; WASP, Wiskott-Aldrich syndrome protein; WIP, WASP-interacting protein; RTK, receptor tyrosine kinase; GST, glutathione S-transferasemAb, monoclonal antibodyPAGE, polyacrylamide gel electrophoresisECL, enhanced chemiluminescent detection.
2 N. M. Quiles, I. M. Antón, N. Ramesh, and R. S. Geha, unpublished results.
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REFERENCES |
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