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• W2024220764 abstract "We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. Src homology polymerase chain reaction glutathioneS-transferase amino acid(s) dorsoanterior index platelet-derived growth factor polyacrylamide gel electrophoresis wild type hemagglutinin fibroblast growth factor Nck is an Src homology (SH)1 2/SH3 adaptor protein, which is thought to mediate the formation of multiprotein complexes in signal transduction. It consists of three SH3 domains and an SH2 domain (1Lehman J.M. Riethmuller G. Johnson J.P. Nucleic Acids Res. 1990; 18: 1048Crossref PubMed Scopus (160) Google Scholar), each of which can bind to other proteins in vivo andin vitro (reviewed in Ref. 2McCarty J.H. BioEssays. 1998; 20: 913-921Crossref PubMed Scopus (80) Google Scholar). Little is known about the physiological role of Nck, although the Drosophila homolog, Dock, has been shown to be required for proper guidance and targeting of retinal axons (3Garrity P.A. Rao Y. Salecker I. McGlade J. Pawson T. Zipursky S.L. Cell. 1996; 85: 639-650Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Nck has also recently been implicated in modulating the response to T cell receptor engagement (4Bubeck Wardenburg J. Pappu R. Bu J.Y. Mayer B. Chernoff J. Straus D. Chan A.C. Immunity. 1998; 9: 607-616Abstract Full Text Full Text PDF PubMed Google Scholar, 5Yablonski D. Kane L.P. Qian D. Weiss A. EMBO J. 1998; 17: 5647-5657Crossref PubMed Scopus (109) Google Scholar) and in actin polymerization on the surface of vaccinia virus particles (6Frischknecht F. Moreau V. Rottger S. Gonfloni S. Reckmann I. Superti-Furga G. Way M. Nature. 1999; 401: 926-929Crossref PubMed Scopus (349) Google Scholar). Earlier work from our laboratory implicated Nck (and Nck-interacting proteins) in mesoderm patterning during early Xenopusdevelopment. We found that injection of RNA encoding mutant forms of Nck in which the first two SH3 domains were intact into the two dorsal blastomeres of four-cell stage embryos resulted in developmental defects. Injected embryos exhibited anterior truncation, and both whole embryos and activin-treated animal cap explants showed evidence of ventralization, with presumptive dorsal mesoderm adopting a more ventral fate (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). The simplest interpretation of these results is that Nck expression alters the response of presumptive dorsal mesoderm to morphogenetic signals, affecting mesoderm patterning prior to and during gastrulation and thereby leading to defects in the dorsoanterior tissues of the resulting embryo. Mutation of the SH2 domain or either of the first two SH3 domains (SH3-1 and SH3-2) of Nck diminished ventralizing activity, while a construct encoding both domains (SH3-1+2) fused to a membrane-targeting signal promoted ventralization (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). We therefore concluded that relocalization of an endogenous protein or proteins that binds both SH3-1 and SH3-2 to sites of tyrosine phosphorylation on the membrane was responsible for the Nck-induced ventralization/anterior truncation phenotype. Here we describe a screen for Xenopus Nck-binding proteins that may function downstream of Nck in mesoderm patterning. The results of this screen suggest that the Cbl proto-oncogene, and Arg and/or its close relative the Abl proto-oncogene, are likely to play a role in the Nck-induced patterning defects. The Abl and Arg nonreceptor tyrosine kinases are implicated in the control of cell proliferation, as well as neural development and responses to adhesion and DNA damage (Refs. 8Kruh G.D. Perego R. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5802-5806Crossref PubMed Scopus (136) Google Scholarand 9Koleske A.J. Gifford A.M. Scott M.L. Nee M. Bronson R.T. Miczek K.A. Baltimore D. Neuron. 1998; 21: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar; reviewed in Refs. 10Van Etten R.A. Trends Cell Biol. 1999; 9: 179-186Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar and 11Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar). Cbl has long been known to down-modulate signals from some tyrosine kinases (12Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (282) Google Scholar), and has recently been shown to be a component of a ubiquitin ligase complex, targeting its tyrosine-phosphorylated binding partners for internalization and/or degradation (13Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar, 14Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 15Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 16Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar). To our surprise, we find that inXenopus Abl family kinases and Cbl work coordinately to enhance the Nck-induced phenotype. Although previous results had suggested that Nck SH3 domains could bind Abl and Cbl (17Wunderlich L. Goher A. Farago A. Downward J. Buday L. Cell Signal. 1999; 11: 253-262Crossref PubMed Scopus (35) Google Scholar, 18Ren R. Ye Z.-S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar, 19Rivero-Lezcano O.M. Sameshima J.H. Marcilla A. Robbins K.C. J. Biol. Chem. 1994; 269: 17363-17366Abstract Full Text PDF PubMed Google Scholar), these data provide the first evidence for the concerted actions of Abl/Arg, Nck, and Cbl in signal transduction. HA-tagged SH3 domains from human Nck were generated by polymerase chain reaction (PCR) (20Lu W. Katz S. Gupta R. Mayer B.J. Curr. Biol. 1997; 7: 85-94Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and cloned into pGEX-2N. Glutathione S-transferase (GST) fusion proteins were purified by binding to glutathione-agarose beads using standard protocols. Full-length human Cbl cDNA was obtained from H. Band. Internal deletion mutants of Cbl and murine Abl were generated by two-step PCR mutagenesis; primer sequences are available upon request. The Xenopus Abl fragment corresponds to amino acids (aa) 110–517 of murine type IV Abl and was kindly provided by K. Dorey and G. Superti-Furga. RNA was purified from stage 8 Xenopus embryos using Trizol reagent (Life Technologies, Inc.), mRNA isolated by oligo(dT) cellulose chromatography, and cDNA was synthesized using oligo(dT) primers and a cDNA synthesis kit (Stratagene). cDNAs were ligated into the Lambda ZAPII vector and packaged following manufacturer's recommendations (Stratagene). Average insert size ranged from 0.5 to 6 kilobase pairs, analyzed by agarose gel electrophoresis. ∼50,000 phage were plated and duplicate nitrocellulose filters obtained. Filters were blocked overnight with 1% ovalbumin in Tris-buffered saline plus Tween 20 (TBST: 150 mm NaCl, 10 mmTris pH 8.0, 0.05% Tween 20) at 4 °C, then incubated in the same buffer containing GST-Nck SH3-1+2-HA (2 μg/ml) for 2 h at 4 °C. Positive plaques were detected with monoclonal anti-HA antibody (HA-11, BAbCo), followed by goat anti-mouse conjugated alkaline phosphatase (Jackson Immunoresearch). False positives were eliminated by binding to GST and detection with a monoclonal anti-GST antibody (21Tanaka S. Morishita T. Hashimoto Y. Hattori S. Nakamura S. Shibuya M. Matuoka K. Takenawa T. Kurata T. Nagashima K. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3443-3447Crossref PubMed Scopus (359) Google Scholar) (kindly provided by M. Matsuda). cDNAs were excised from positive clones with the ExAssist helper phage (Stratagene). TheXenopus Arg and Cbl phage clones correspond to aa 392–end of human type Ib Arg and aa 342–end of human Cbl, respectively. The sequences have been deposited in GenBank™ (accession numbers AF237765and AF237766). Partial cDNAs were transcribed and translated in the presence of [35S]methionine using the TnT Quick Coupled Transcription/Translation System following manufacturer's protocols (Promega). One third of the reaction contents were then incubated with 2.5 μg of GST proteins bound to 5 μl of glutathione-agarose beads in 75 μl of TNGT (20 mm Tris, pH 7.4, 150 mm NaCl, 10% glycerol, 0.1% Triton X-100) at 4 °C for 1 h. Beads were washed four times with TNGT, and bound proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RNA was prepared from pooled staged embryos using Trizol reagent following manufacturer's instructions. Polyadenylated RNA was isolated by oligo(dT) chromatography using standard protocols. One μg of poly(A)+ RNA/lane was electrophoresed in agarose gels containing formaldehyde and transferred to nylon membranes by capillary action. Filters were hybridized with [α-32P]UTP-labeled riboprobe overnight and washed three times in 0.1× SSPE (1× SSPE: 150 mm NaCl, 10 mm NaH2PO4, 1.25 mmEDTA, pH 7.4), 0.5% SDS at 65 °C before exposure to film. Antisense RNA probes were synthesized in vitro using T7 or T3 RNA polymerase from linearized pBK (Stratagene) or pGHXP (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar) plasmid templates. Human 293T cells were maintained and transfected as described (22Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar) and lysed in 1 ml of TXB (20 mm Tris, pH 7.4, 150 mmNaCl, 5 mm EDTA, 1% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 1% (v/v) aprotinin solution (Sigma A6279)). For Fig. 3 B, 300 μl of normalized lysate was precleared with 30 μl of protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. Supernatants were then incubated with 1 μg of affinity-purified rabbit anti-Nck antibody or 1 μl of crude preimmune serum for 2 h at 4 °C. Immune complexes were collected on 20 μl of protein A beads and washed three times in Tris-buffered saline plus Tween 20 before adding sample buffer, boiling, and subjecting to SDS-PAGE and immunoblotting with monoclonal anti-Abl antibody (8E9, PharMingen). For Fig. 4, 100 μl of lysate was pre-cleared for 1 h with 10 μl of protein G-agarose beads (Pierce) and supernatant incubated with 1 μg of 9E10 anti-Myc monoclonal antibody for 2 h at 4 °C. Immunoprecipitates were collected on 10 μl of protein G-agarose beads and washed twice in TXB, once in TXB with 0.5 m NaCl, once in TXB with 10 mm NaCl, and twice in TXB again before SDS-PAGE and immunoblotting with polyclonal anti-HA antibody.Figure 4Mapping the binding site for Nck in Cbl.Full-length human Nck (tagged with Myc epitope) and Cbl mutants (tagged with HA epitope) were cotransfected as indicated in 293T cells. Nck was immunoprecipitated with anti-Myc antibody and immunoprecipitates subjected to immunoblotting with anti-HA to detect Cbl (top). Bottom panel, whole cell lysates immunoblotted with anti-HA to detect Cbl. Mutant 480 has a deletion of aa 481–end; ΔPRD-1 lacks residues 483–688; ΔPRD-2 lacks residues 820–826; ΔPRD-1+2 has both deletions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All constructs used to synthesize mRNA for microinjection were in the vector pGHXP, which is derived from pGEM-HE (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). Nck mutants used here have been described (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). Murine type IV c-Abl, human type 1B Arg (provided by G. Kruh), and HA-tagged human c-Cbl were fitted with appropriate restriction sites by PCR and subcloned into pGHXP. After linearization of plasmids withNheI, capped mRNAs were synthesized by in vitro transcription using the mMessage mMachine T7 kit (Ambion, Inc.) following manufacturer's protocols. RNA was quantitated by ethidium bromide staining. Frogs were maintained and fertilized eggs obtained following standard protocols (23Newport J. Kirschner M. Cell. 1982; 30: 675-686Abstract Full Text PDF PubMed Scopus (1183) Google Scholar). Embryo staging and quantitation of dorsoanterior index (DAI) were according to Nieuwkoop and Faber (24Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis. North-Holland, Amsterdam1967Google Scholar) and Kao and Elinson (25Kao K.R. Elinson R.P. Dev. Biol. 1988; 127: 64-77Crossref PubMed Scopus (423) Google Scholar), respectively. Our previous experiments involving expression of Nck mutants in early Xenopus embryos showed that Nck constructs in which the first and second SH3 domains (SH3-1 and SH3-2) were intact induced anterior truncation and mesoderm ventralization (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). Because Nck consists only of modular protein-interaction domains, this phenotype must be a consequence of endogenous proteins binding to Nck. Since our prior mutational analysis suggested that both SH3 domains of Nck were required in cis for the mesoderm patterning phenotype (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar), we aimed to identify Xenopus proteins that preferentially bind to Nck SH3-1+2 compared with either domain alone. We constructed a λ phage expression library using cDNA from mid-blastula (stage 8) embryos and screened the library for binding to a GST-SH3-1+2 fusion protein. Thirty-nine independent SH3-1+2-binding clones were isolated and further characterized. We tested each of these clones for binding to purified SH3 domains, and only two of these were found to bind more tightly to SH3-1+2 than to the individual SH3 domains. Sequencing revealed that these clones corresponded to theXenopus homologs of the proto-oncogene Cbl and the nonreceptor tyrosine kinase Arg, which is highly related to the Abl proto-oncogene. As shown in Fig. 1, when the cDNA inserts corresponding to Xenopus Arg and Cbl were transcribed and translated in vitro, the translation products bound very well to the GST fusion containing both SH3-1 and SH3-2, but weakly or not at all to the individual domains. This is consistent with results of another group, which found that multiple SH3 domains were required for mammalian Nck to interact with Cbl or BCR-Abl (17Wunderlich L. Goher A. Farago A. Downward J. Buday L. Cell Signal. 1999; 11: 253-262Crossref PubMed Scopus (35) Google Scholar). For all other clones, solution binding was either weak for all domains tested, or required only the second SH3 domain, which seems to dominate interactions relative to SH3-1 (data not shown). Because the mesoderm patterning defect associated with Nck expression is first manifested in the early gastrula stage (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar), anyXenopus protein involved in the phenotype would need to be present by this stage of development. To confirm that Arg and Cbl were expressed at this time, we performed Northern blots of poly(A)+ RNA from staged embryos probed with theXenopus Arg and Cbl probes. As seen in Fig.2, both messages were present in the unfertilized egg and throughout the cleavage and blastula stages. In the case of Arg, there was a spike of expression at stage 8 (corresponding to the mid-blastula transition, where zygotic expression begins) followed by a rapid decline after gastrulation. For Cbl, there was a large store of maternal message and relatively constant expression throughout development compared with the ornithine decarboxylase control message. We also obtained a cDNA forXenopus Abl (which is highly related to Arg) and examined its expression. In this case the pattern was more complex, showing several spliced forms or related messages, but there was significant expression of all forms throughout early development. We performed whole mount in situ hybridization experiments to localize Arg and Cbl message. In both cases, diffuse staining was observed from the late blastula through mid-gastrula stages; this staining was strongest near the animal pole, with no obvious dorsoventral bias (data not shown). We have previously shown that endogenous Nck message is present in maternal stores and is induced at the mid-blastula transition (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). Therefore Arg, Abl, and Cbl are all expressed in the blastula and early gastrula stages, consistent with a role in the Nck-induced mesoderm patterning defect. We next mapped the binding sites for Nck SH3 domains on Cbl and Abl. We chose to map the sites in human Cbl and murine Abl and not theXenopus Cbl and Arg clones for several reasons. First, we had full-length cDNAs and a variety of mutants for the mammalian homologs available. More importantly, we wish ultimately to relate the results from Xenopus injection experiments to mammalian systems, where both Cbl and Abl have been shown to function as oncogenes and otherwise affect signaling. Because theXenopus and mammalian versions of Cbl and Arg are so similar (66% and 64% identity for Cbl and Arg, respectively), it is reasonable to assume that their function will be conserved across species. Human Abl and Arg share over 90% identity in the region spanning the first common exon, SH3, SH2, and catalytic domains (8Kruh G.D. Perego R. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5802-5806Crossref PubMed Scopus (136) Google Scholar); although Abl and Arg differ somewhat in their biological activities (26Wang B. Kruh G.D. Oncogene. 1996; 13: 193-197PubMed Google Scholar, 27Mysliwiec T. Perego R. Kruh G.D. Oncogene. 1996; 12: 631-640PubMed Google Scholar), presumably due to their fairly divergent C termini, mouse knockout experiments have shown that they are at least in part functionally redundant (9Koleske A.J. Gifford A.M. Scott M.L. Nee M. Bronson R.T. Miczek K.A. Baltimore D. Neuron. 1998; 21: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). To map the binding sites in Abl for Nck SH3-1+2, we synthesized the catalytic domain and C terminus of c-Abl and various deletion mutants by in vitro transcription/translation and tested their ability to bind to GST-SH3-1+2 on beads. As shown in Fig.3 A, deletion of a proline-rich region of the Abl C terminus adjacent to the catalytic domain (ΔPro) eliminated binding to SH3-1+2; this region contains two binding sites for Crk SH3 domains, a nuclear localization signal, and a canonical type 2 SH3-binding site (18Ren R. Ye Z.-S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar, 28Van Etten R.A. Jackson P. Baltimore D. Cell. 1989; 58: 669-678Abstract Full Text PDF PubMed Scopus (336) Google Scholar, 29Feller S.M. Knudsen B. Hanafusa H. EMBO J. 1994; 13: 2341-2351Crossref PubMed Scopus (326) Google Scholar). Deletion of the two Crk binding sites (“Crkless”) did not affect Nck binding, whereas deletion of the third site (“Nckless”) essentially eliminated binding of Abl to SH3-1+2. These experiments confirm that the predominant binding site in Abl for Nck SH3 domains is a single PXXP at position 631–636, as suggested previously (18Ren R. Ye Z.-S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar). This site is conserved betweenXenopus Arg (APQPPKR, with canonical type 2 SH3-binding site residues underlined), human Arg, and human and mouse Abl (APTPPKR), and Drosophila Abl (APAPPKR). We also examined the association of Nck with Abl in vivo. In lysates of 293T cells overexpressing full-length Nck and wt Abl, Abl efficiently co-immunoprecipitated with Nck (Fig. 3 B). Mutants lacking the proline-rich Nck SH3 domain binding site also coprecipitated with Nck, albeit at somewhat lower levels. We have previously shown that binding of a Nck SH3-1+2 construct to Abl in 293T cells was almost completely eliminated by deletion of the proline-rich region of Abl (30Smith J.M. Katz S. Mayer B.J. J. Biol. Chem. 1999; 274: 27956-27962Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar); on the other hand, in experiments in cells expressing full-length Nck constructs, less Abl was co-precipitated with Nck SH2 domain mutants than with wt Nck. 2T. Miyoshi-Akiyama, L. M. Aleman, J. M. Smith, C. E. Adler, and B. J. Mayer, submitted for publication. Taken together, these results suggest that, in addition to the Nck SH3-mediated interaction mapped here, the SH2 domain of Nck can also contribute to Abl bindingin vivo. The Nck binding sites in human Cbl were identified by co-expression of full-length Myc epitope-tagged Nck with HA epitope-tagged Cbl in human 293T cells. Nck was immunoprecipitated with anti-Myc antibody, and coprecipitating Cbl was identified by immunoblotting with anti-HA antibody. As shown in Fig. 4, deletion of the entire C terminus of Cbl (mutant 480) virtually abolished Nck binding. However, deletion of the major proline-rich region of the C terminus between amino acids 483 and 688 (ΔPRD1) did not affect binding, nor did deletion of a potential type 2 SH3-binding site at amino acids 820–826 (ΔPRD2). However when these two deletions were combined in mutant ΔPRD1+2, binding to Nck was almost completely eliminated, suggesting that either of these two regions is sufficient for binding to Nck SH3 domains. Although the SH2 and SH3-3 domains are also present in wt Nck, under the conditions of this experiment, they have only a modest effect on Cbl binding.2 The C-terminal Nck-binding site that we identified (VPERPPK) is absolutely conserved in mouse, human, and Xenopus Cbl, but is not present in Cbl-b or Drosophila Cbl; a potential variant is found inCaenorhabditis elegans Sli-1 (VPLPPAR). Our major goal was to ascertain whether either Abl (or Arg) or Cbl was involved in the anterior truncation/ventralization phenotype associated with Nck overexpression in Xenopus. Our previous results suggested that Nck mutants exerted their ventralizing effect by recruiting Xenopus proteins to the membrane (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). We therefore reasoned that constitutive localization of Abl, Arg, or Cbl should ventralize in the absence of Nck mutant expression if it were the critical effector. It was impossible to test the effect of direct membrane localization of Abl and Arg, as both proteins already contain an N-terminal membrane localization signal (8Kruh G.D. Perego R. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5802-5806Crossref PubMed Scopus (136) Google Scholar, 31Ben-Neriah Y. Bernards A. Paskind M. Daley G.Q. Baltimore D. Cell. 1986; 44: 577-586Abstract Full Text PDF PubMed Scopus (171) Google Scholar) and other targeting sequences (e.g. actin-binding and nuclear localization signals), suggesting that the protein is normally partitioned among several subcellular compartments. In the case of Cbl, we constructed a version containing an N-terminal myristoylation signal derived from Src and tested the effects of expression by injecting mRNA into the two dorsal blastomeres of four-cell stage embryos. No obvious phenotypic effects were observed (data not shown), suggesting that relocalization of Cbl to the membrane was unlikely to be uniquely responsible for the Nck-induced phenotype. We also reasoned that if binding of Nck to endogenous Abl, Arg, or Cbl was responsible for the phenotype, overexpression of these proteins should enhance the ventralizing effect of Nck. The Lys-229 mutant of Nck, in which SH3-3 is mutated but all other domains are intact, is the most potent Nck mutant for anterior truncation/mesoderm ventralization (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar). When Nck Lys-229 is expressed at low levels (100 pg of RNA/embryo), however, the great majority of embryos develop normally (Table I and Fig.5). This sensitized system allowed us to test the effects of overexpression of candidate Nck effectors. As shown in Table I and Fig. 5, overexpression of Abl or Cbl alone did not enhance anterior truncation by Nck Lys-229. However, the combination of Nck Lys-229, Abl, and Cbl induced a phenotype that was much stronger than that induced by injection of any of the RNAs individually or pairwise. The effect of wt Nck, which we had previously shown to be less potent than the Lys-229 mutant (7Tanaka M. Lu W. Gupta R. Mayer B.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4493-4498Crossref PubMed Scopus (30) Google Scholar), was also enhanced by the combination of Abl and Cbl (Table I). We eliminated the possibility that this phenotype was simply due to increased levels of injected RNA, as neither co-injection of Abl and Cbl with a Nck mutant in which all binding domains are mutated (Nck Kall), nor co-injection of Nck Lys-229 and Abl with a Cbl mutant that cannot bind Nck (Cbl Δ1+2), could induce anterior truncation (Table I). Finally, human Arg behaved identically to Abl in these assays, synergizing with Cbl and Nck Lys-229 (Table I). Together, these results imply that anterior truncation/ventralization induced by Nck involves both Cbl and Abl family kinases.Table IInjected mRNAsmRNA injectedpg RNA/embryoDAINo. injected (% survival)54–32–1Experiment 1 Lys-22910096.63.40140 (96.7) Cbl1001000050 (96.0) Abl5089.110.9075 (73.3) Lys-229 + Abl100 + 507525050 (64.0) Abl + Cbl50 + 10080.919.1075 (90.7) Lys-229 + Cbl100 + 1001000075 (97.3) Lys-229 + Abl + Cbl100 + 50 + 10045.742.911.450 (96.0)Experiment 2 Lys-229 + Abl + Cbl100 + 50 + 10024.371.44.375 (93.3) wt Nck + Abl + Cbl100 + 50 + 1004750375 (88.0) Nck Kall + Abl + Cbl100 + 50 + 10080.617.91.575 (89.3) Lys-229 + Abl + CblΔ(1 + 2)100 + 50 + 1008218075 (97.3)Experiment 3 Arg5093.35.31.375 (100) Arg + Cbl50 + 10097.32.7075 (90.1) Lys-229 + Arg50 + 10076.720.12.975 (100) Lys-229 + Arg + Cbl50 + 100 + 10049.242.38.675 (94.7)mRNAs indicated were injected into the two dorsal blastomeres of four-cell-stage embryos and DAI was scored at stage 35. DAI 5 embryos are normal, whereas smaller numbers indicate increasing dorsoanterior deficiencies (22Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar). Representative data from several experiments are presented; all experiments were repeated several times with essentially similar results." @default.
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