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• W2074401103 abstract "N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center. N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center. Cell migration is an important biological process in embryonic development as well as wound repair, angiogenesis, and tumor metastasis (1Christopher R.A. Guan J.L. Int. J. Mol. Med. 2000; 5: 575-581PubMed Google Scholar, 2Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3276) Google Scholar). Cell migration is a multistep process involving protrusion of the cell membrane and formation of a new attachment in the leading edge, myosin/actin-mediated cell contraction and release of attachment at the rear part of the cell. When they first contact the substrate, cells spread to maximize the contact area by protruding the membrane, which is considered to be similar to the forward extension of filopodia and lamellepodia in the initial step of cell migration (3Wakatsuki T. Wysolmerski R.B. Elson E.L. J. Cell Sci. 2003; 116: 1617-1625Crossref PubMed Scopus (129) Google Scholar). Cell spreading is driven by actin polymerization, which is regulated by actin nucleation machinery involving Arp2/3 complex (4Welch M.D. Trends Cell Biol. 1999; 9: 423-427Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Arp2/3 complex is activated by the Wiskott-Aldrich syndrome protein (WASP) 2The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; hnRNPK, heterogeneous nuclear ribonucleoprotein K; VCA, verproline homology, cofilin homology, acidic; KH, K homology; KI, K interaction; SIC, spreading initiation center; HA, hemagglutinin; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; GFP, green fluorescent protein; FN, fibronectin; MEF, mouse embryonic fibroblast; FAK, focal adhesion kinase; WCL, whole cell lysates.2The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; hnRNPK, heterogeneous nuclear ribonucleoprotein K; VCA, verproline homology, cofilin homology, acidic; KH, K homology; KI, K interaction; SIC, spreading initiation center; HA, hemagglutinin; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; GFP, green fluorescent protein; FN, fibronectin; MEF, mouse embryonic fibroblast; FAK, focal adhesion kinase; WCL, whole cell lysates. protein family through direct binding to the conserved verproline homology, cofilin homology, acidic (VCA) domain of WASP (5Rohatgi R. 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Cell. 2004; 117: 649-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), using a proteomic approach, discovered that hnRNPK is also involved in cell adhesion. They showed that hnRNPK is present in a novel structure called the spreading initiation center (SIC), which is similar, but distinct, from the more mature focal adhesions. A functional role of hnRNPK in cell adhesion is supported by the observation that inhibition of hnRNPK by antibodies led to an increased spreading of the cell. This study suggests a potential role of hnRNPK in cell spreading; nevertheless, little is known about the mechanism by which hnRNPK regulates cell spreading. In this study, we identify a direct interaction between N-WASP and hnRNPK, which is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its KI domain. We found that N-WASP and hnRNPK are co-localized at the cell periphery, which resembles the SIC, in the early spreading stage. Furthermore, co-expression of hnRNPK reverses N-WASP-induced filopodia formation and cell spreading. These results suggest that hnRNPK regulates cell spreading through its inhibition of N-WASP. Antibodies—The rabbit polyclonal α-HA (Y11) antibody, the mouse monoclonal α-c-Myc-tag (9E10) antibody, the rabbit polyclonal α-hnRNPK antibody, and the rabbit polyclonal α-ParP antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Affinity-purified antibody against GST was prepared from anti-GST serum using GST immobilized on glutathione-Sepharose as an affinity matrix. The mouse monoclonal α-vinculin antibody was obtained from Sigma. The rabbit antibody against N-WASP was a generous gift of Dr. H. Miki (University of Tokyo). Cell Culture and Transfection—293 cells were cultured in DMEM with 10% FBS. mouse embryonic fibroblast (MEF) cells derived from mouse embryo were maintained in DMEM supplemented with 10% FBS. CHO cells were cultured in Ham's F-12 with 10% FBS, and NIH3T3 cells were maintained in DMEM with 10% calf serum. Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's guidelines. Transfection efficiency was about 90% in 293T cells, 10–20% in NIH3T3 cells and 40–50% in CHO cells as detected by fluorescent microscopy using GFP as a marker. In case of co-transfection, each plasmid encoding protein was used in a 1:1 ratio. Co-transfection efficiency was determined to be nearly 100% by indirect immunofluorescent staining using anti-Myc and anti-HA. In some experiments, GFP-encoding plasmid was used as transfection marker. The ratio between GFP, HA-tagged and Myc-tagged protein-coding plasmid was 1:3:3. After transfection, GFP-positive cells were considered as co-transfected cells. Plasmid Construction—pKH3-N-WASP, pHAN-N-WASP, pDHGST-N-WASP, pDH-N-WASP1, pDH-N-WASP2, pDH-N-WASP3, pDH-N-WASP4, and pEGFP-N-WASP have been described previously (42Wu X. Suetsugu S. Cooper L.A. Takenawa T. Guan J.L. J. Biol. Chem. 2004; 279: 9565-9576Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The hnRNPK cDNA, a generous gift from Dr. David Levens (National Institutes of Health), was used as a template for PCR amplification. The PCR product with the primers 5′-tcagatgaattcatatggaaactgaacagccagaagaaaccttc-3′ and 5′-taaagcgaattctaagaaaaactttccagaatactgcttcac-3′ was digested with EcoRI and then ligated to a linearized pKH3 vector with EcoRI sites on the ends to generate pKH3-hnRNPK. The EcoRI fragment digested from pKH3-hnRNPK was inserted into linearized pHAN (43Han D.C. Shen T.L. Miao H. Wang B. Guan J.L. J. Biol. Chem. 2002; 277: 45655-45661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), pGEX2T at the corresponding site to generate pHAN-hnRNPK and pGEX2T-hnRNPK, respectively. hnRNPK truncation mutant encoding residues 1–337(K1), 1–209(K2), 1–104(K3), and 171–337(K4) were made by PCR using primers: 5′-ttcaggatccccgggaatggaaactgaacagccagaagaaaccttc-3′(hnRNPK-5) and 5′-taaagggaattcattaaaccatgccgtcgtaacggtctccaggtct-3′ (337-3), hnRNPK-5 and 5′-taaagggaattcattagatgatctttatgcactctacaaccctatc-3′ (209-3), hnRNPK-5 and 5′-taaagggaattcattagattttcttcagaatttctccaattgtttc (104-3), 5′-ttcaggatccccgggacgagagaacactcaaaccaccatcaagctt-3′ (171-5) and 337-3, respectively. The PCR products were digested with SmaI and EcoRI and then inserted into pKH3 at the corresponding sites to generate pKH3-K1, pKH3-K2, pKH3-K3, and pKH3-K4, respectively. Immunoprecipitation and Western Blotting—Subconfluent cells were washed with ice-cold phosphate-buffered saline twice and lysed with 1% Nonidet P-40 lysis buffer (20 mm Tris, pH 8.0, 137 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm NaVO4, 1 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinine, and 20 mg/ml leupeptin). Lysates were cleared by centrifugation for 20 min at 4 °C, and protein concentration was determined by Bio-Rad protein assay. Immunoprecipitations were performed by incubating cell lysates with appropriate antibodies, as indicated, for more than 2 h at 4 °C. For the experiment to detect interaction between endogenous proteins, Immunoprecipitations were followed by incubation with protein A-Sepharose for another 2 h. After three times washing, the immune complex was resolved by SDS-PAGE. Western blotting was carried out using horseradish peroxidase-conjugated IgG as a secondary antibody and ECL system for detection. Preparation of GST Fusion Proteins and in Vitro Binding Assay—GST fusion proteins were produced and purified as described previously (44Reiske H.R. Kao S.C. Cary L.A. Guan J.L. Lai J.F. Chen H.C. J. Biol. Chem. 1999; 274: 12361-12366Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). GST fusion proteins were immobilized on glutathione-agarose beads and then incubated with recombinant His-tagged N-WASP for more than 2 h at 4°C in 300 μl of Nonidet P-40 buffer. After washing, the bound proteins were analyzed by Western blotting with antibody for N-WASP. For in vitro binding assays to measure the binding constant, a serially increasing amount of His-tagged N-WASP (0.003–0.2 μm) and GST-hnRNPK or GST-K4 fragment were used. The intensity of the bands was quantified with Image J software. To calculate the binding constant (Kd), the resulting data were fit to a single rectangular hyperbola equation with Prism 3.0 (Graph Pad Software, San Diego, CA): B = BmaxC/(Kd + C), where B is the relative value of bound protein, and C is the concentration of samples tested. Nuclear and Cytoplasmic Fractionation—Fractionation was performed essentially as described (45Lin S.Y. Makino K. Xia W. Matin A. Wen Y. Kwong K.Y. Bourguignon L. Hung M.C. Nat. Cell Biol. 2001; 3: 802-808Crossref PubMed Scopus (892) Google Scholar). Briefly, cells were lifted by trypsinization, washed with phosphate-buffered saline, then lysed in a lysis buffer (20 mm Hepes, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.5% Nonidet P-40, 1mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 20 mg/ml leupeptin) for 10 min. The lysates were centrifuged at 1,500 × g for 5 min to sediment nuclei. The supernatant was then centrifuged at 15,000 × g for 10 min, and the supernatant formed the cytoplasmic fraction. The nuclear pellet was washed three times with lysis buffer and then resuspended in the same lysis buffer supplemented with 0.5 m NaCl to extract nuclear proteins. The extracted nuclear proteins were sedimented at 15,000 × g for 10 min, and the resulting supplement was harvested as the nuclear fraction. Fluorescent Microscopy—Cells were processed for immunofluorescent staining as described previously (46Zhao J.H. Reiske H. Guan J.L. J. Cell Biol. 1998; 143: 1997-2008Crossref PubMed Scopus (300) Google Scholar). Cells were lifted by trypsinization, pelleted, resuspended in serum-free media, and incubated at 37 °C for 1 h with gentle agitation. The cells were pelleted, resuspended in media with 10% FBS, and then allowed to spread on the fibronectin (FN)-coated coverslip. After 25 min, cells were fixed with 3.7% formaldehyde and subjected to fluorescent immunostaining. The primary antibodies were rabbit anti-HA antibody and mouse monoclonal anti-Myc antibody. For secondary antibodies, Texas Red-conjugated goat anti-rabbit antibody (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA) and fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody were used. For confocal microscopy, coverslips were imaged using Leica TCS SP2 (sequential scan). For phalloidin staining to detect filopodia formation, cells in suspension were pelleted, resuspended in media with 0.2% calf serum, and then allowed to spread on the FN-coated coverslip. 30 min after plating, cells were fixed and subject to immunofluorescent staining. GFP was observed directly by fluorescent microscopy. Cell Spreading Assay—Cell spreading assays were performed as described previously (47Han D.C. Rodriguez L.G. Guan J.L. Oncogene. 2001; 20: 346-357Crossref PubMed Scopus (82) Google Scholar). Briefly, cells were lifted by trypsinization, pelleted, resuspended in serum-free media and incubated at 37 °C for 1 h with gentle agitation. The cells were pelleted, resuspended in media with 0.2% FBS, and then allowed to spread on the FN-coated plate. After 1.5 h, cells were fixed with 3.7% formaldehyde and photographed. Spread cells were defined as cells with irregular morphology and lacking phase brightness; non-spread cells were rounded and phase-bright under the microscope. Multiple fields were imaged and ∼200 transfected cells were monitored and counted blindly for each experiment. Three independent experiments were performed, and the Student's t test was used to determine the statistical significance. Pyrenyl Actin Polymerization Assays—Bovine Arp2/3 complex 10 nm and activator were added to 1.5 μm Mg2+-ATP-G-actin (10% pyrene labeled) in KMET buffer (50 mm KCl, 1 mm MgCl2, 1 mm EGTA, 10 mm Tris, pH 7.0) supplemented with 0.5 mm ATP. Actin polymerization was monitored by continuous pyrene fluorescence measurements (λex = 386 nm, λem = 407 nm) in a Cary Eclipse fluorescence spectrophotometer (Varian). Actin was purified from rabbit muscle and isolated as Ca2+-ATP-G-actin in G buffer (5 mm Tris-Cl, pH 7.8, 0.1 mm CaCl2, 0.2 mm ATP, and 1 mm dithiothreitol) according to Pardee and Spudich (48Pardee J.D. Spudich J.A. Methods Cell Biol. 1982; 24: 271-289Crossref PubMed Scopus (339) Google Scholar) and pyrenyl labeled. Bovine GST-N-WASP WA and bovine Arp2/3 complex were purified as described previously (49Egile C. Loisel T.P. Laurent V. Li R. Pantaloni D. Sansonetti P.J. Carlier M.F. J. Cell Biol. 1999; 146: 1319-1332Crossref PubMed Scopus (428) Google Scholar). Identification of N-WASP Interaction with hnRNPK—To further investigate the potential mechanisms of regulation of N-WASP, we searched for additional proteins that interact with N-WASP via tandem tag affinity purification followed by mass spectrometry for protein identification. One of the novel N-WASP-associated proteins was identified as an RNA-binding protein, hnRNPK. Although hnRNPK was not known to be involved in actin-related cellular function, de Hoog et al. (41de Hoog C.L. Foster L.J. Mann M. Cell. 2004; 117: 649-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) recently reported that hnRNPK is localized in the SIC and the inhibition of hnRNPK by antibodies led to an increased cell spreading, suggesting a potential role for hnRNPK in the down-regulation of cell adhesion. To validate association of N-WASP with hnRNPK, 293 cells were co-transfected with plasmids encoding Myc-tagged N-WASP and HA-tagged hnRNPK or HA alone as a control. The cell lysates were then immunoprecipitated with antibody against HA and blotted with anti-Myc. Fig. 1A shows that N-WASP was associated with hnRNPK but not present in the immunoprecipitates from control cells. To examine the interaction between endogenous N-WASP and hnRNPK, co-immunoprecipitation experiments were performed using cell lysates prepared from NIH3T3 cells. The lysates were immunoprecipitated by antibodies against N-WASP, and the immune complexes were subjected to Western blotting with anti-hnRNPK to detect associated hnRNPK. Fig. 1B shows the co-precipitation of hnRNPK with N-WASP but not with the control antibody. To determine whether the interaction between N-WASP and hnRNPK is direct or not, we prepared a GST fusion protein containing hnRNPK and a recombinant His-tagged N-WASP protein and used these for an in vitro binding assay. Fig. 1C shows the association of recombinant His-tagged N-WASP with GST-hnRNPK but not with control GST alone. Together, these results identify a specific and direct interaction between N-WASP and hnRNPK. To determine which domain(s) of N-WASP is responsible for binding to hnRNPK, 293 cells were co-transfected with plasmids encoding Myc-tagged hnRNPK and vectors encoding GST fusion proteins containing N-WASP fragments (see Fig. 2A). Lysates were prepared from the transfected cells, and GST fusion proteins were pulled down with glutathioneagarose beads followed by Western blotting with anti-Myc antibody to detect associated Myc-hnRNPK. Fig. 2B shows that hnRNPK was associated with the N-terminal fragment of N-WASP containing EVH1/WH1 domain (residues 1–148) but not with other fragments corresponding to G protein-binding domain, proline-rich, and VCA domains. A similar strategy was used to define the N-WASP binding site on hnRNPK by co-transfection of 293 cells with plasmids encoding Myc-tagged N-WASP and vectors encoding HA-tagged hnRNPK or its fragments (see Fig. 2C). Fig. 2D shows that the full-length hnRNPK, the K1 (residues 1–337) and K4 (residues 171–337, which contains KI domain) fragments were associated with N-WASP, but K2 (residues 1–209) and K3 (residues 1–104) fragments were not. Together, these results suggest that the interaction between N-WASP and hnRNPK is mediated by the EVH1/WH1 domain of N-WASP and the region of hnRNPK containing its KI domain. To obtain the binding constant for the interaction between N-WASP and hnRNPK, an in vitro binding assay using a serially increasing amount of recombinant His-tagged N-WASP and GST-hnRNPK was performed as described under “Experimental Procedures.” The GST pulldown assay was followed by Western blotting with anti-N-WASP antibody, and the intensity of each band was measured by densitometry. We estimated that the binding constant (Kd) is 60 nm. (Fig. 2E). Similar assays showed an approximate Kd of 57 nm for association of N-WASP and hnRNPK K4 fragment (Fig. 2E). These results suggested that the K4 fragment of hnRNPK is primarily responsible for hnRNPK interaction with N-WASP. Co-localization of N-WASP and hnRNPK in the SIC during Early Stage of Cell Spreading—Consistent with its role in the regulation of actin polymerization, N-WASP is localized in the cell periphery (42Wu X. Suetsugu S. Cooper L.A. Takenawa T. Guan J.L. J. Biol. Chem. 2004; 279: 9565-9576Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Interestingly, recent studies suggested that hnRNPK is localized in the SIC, a distinctive patch-like structure associated with the cell periphery, in the early stage of cell spreading (41de Hoog C.L. Foster L.J. Mann M. Cell. 2004; 117: 649-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Thus, we examined potential co-localization of N-WASP and hnRNPK in cells during their early spreading stage. Primary MEFs were co-transfected with plasmids encoding HA-tagged hnRNPK and Myc-tagged N-WASP or a control irrelevant protein, GST. Transfected cells were suspended, and replated on FN-coated coverslips. They were fixed in the early spreading stage and subjected to double-label immunofluorescence using rabbit anti-HA and mouse anti-Myc antibody. Fig. 3 shows that hnRNPK is localized in a distinct circular patch in the cell periphery resembling the SIC described previously (41de Hoog C.L. Foster L.J. Mann M. Cell. 2004; 117: 649-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). N-WASP is co-localized with hnRNPK in a punctuated pattern in the cell periphery, whereas GST is uniformly distributed in the cytoplasm. These results suggest specific interaction between N-WASP and hnRNPK in the SIC in the early spreading stage of the cell. Inhibition of N-WASP Promoted Cell Spreading by hnRNPK—N-WASP is a well established critical regulator of actin polymerization, which is important for filopodia formation and cell spreading. Interestingly, recent studies also suggested a potential role for hnRNPK in the regulation of cell spreading, although the potential mechanisms involved are unknown (41de Hoog C.L. Foster L.J. Mann M. Cell. 2004; 117: 649-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). To determine whether hnRNPK may influence cell spreading through its interaction with N-WASP, CHO cells were transfected with plasmids encoding Myc-tagged N-WASP or HA-tagged hnRNPK, or both plasmids, along with a vector encoding GFP as a transfection marker. Cell spreading was then assessed for transfected cells after re-plating on FN, as described previously (47Han D.C. Rodriguez L.G. Guan J.L. Oncogene. 2001; 20: 346-357Crossref PubMed Scopus (82) Google Scholar). Fig. 4A shows that expression of N-WASP promoted cell spreading a" @default.
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• W2074401103 date "2006-06-01" @default.
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• W2074401103 title "Interaction of N-WASP with hnRNPK and Its Role in Filopodia Formation and Cell Spreading" @default.
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