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• W2046083984 abstract "Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (αI) SH3 domain as a bait. Five isoforms of the αI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two αI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 → p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene14, 233–241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin αIΣI/βIΣI antibody in untransfected NIH3T3 cells; in addition, the anti-αIΣI/βIΣI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against αIΣI/βIΣI spectrin and Abl tyrosine kinase but not against αII/βII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed. Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (αI) SH3 domain as a bait. Five isoforms of the αI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two αI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 → p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene14, 233–241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin αIΣI/βIΣI antibody in untransfected NIH3T3 cells; in addition, the anti-αIΣI/βIΣI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against αIΣI/βIΣI spectrin and Abl tyrosine kinase but not against αII/βII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed. Erythroid spectrin is the predominant component of the two-dimensional protein network called the membrane skeleton, underlying the lipid bilayer of red cells (for recent reviews, see Refs. 1Bennett V. Gilligan D.M. Annu. Rev. Cell Biol. 1993; 9: 27-66Crossref PubMed Scopus (444) Google Scholar, 2Morrow J.S. Rimm D.L. Kennedy S.P. Cianci C.D. Sinard J.H. Weed S.A. Hoffman J. Jamieson J. Handbook of Physiology. Oxford, London1996: 485-540Google Scholar, 3Goodman S.R. Zimmer W.E. Clark M.B. Zagon I.S. Barker J.E. Bloom M.L. Brain Res. Bull. 1995; 36: 593-606Crossref PubMed Scopus (124) Google Scholar). Formation of the membrane skeleton involves multiple protein-protein interactions among integral membrane proteins. Interactions of spectrin with other membrane proteins such as ankyrin, protein 4.1, and adducin provide a linkage of spectrin either to the plasma membrane or among spectrin tetramers. Many hereditary anemia mutations affect interactions of these integral membrane proteins, resulting in increased fragility and shortened lifespan of erythrocytes. In hereditary elliptocytosis and pyropoikilocytosis, the mutations have been localized in the α- and β-subunits of spectrin (reviewed in Refs. 4Lux S.E. Palek J. Handlin R.I. Lux S.E. Stossel T.P. Blood, Principles and Practice of Hematology. J. P. Lippincott Co., Philadelphia1995: 1701-1818Google Scholar and 5Gallagher P.G. Forget B.G. Semin. Hematol. 1993; 30: 4-20PubMed Google Scholar). Many of these proteins, including spectrin, which were first identified in red cells, have isoforms expressed in nonerythroid cells, but the structure and regulatory processes of the nonerythroid membrane skeleton are less well understood (reviewed in Refs. 1Bennett V. Gilligan D.M. Annu. Rev. Cell Biol. 1993; 9: 27-66Crossref PubMed Scopus (444) Google Scholar, 2Morrow J.S. Rimm D.L. Kennedy S.P. Cianci C.D. Sinard J.H. Weed S.A. Hoffman J. Jamieson J. Handbook of Physiology. Oxford, London1996: 485-540Google Scholar, 3Goodman S.R. Zimmer W.E. Clark M.B. Zagon I.S. Barker J.E. Bloom M.L. Brain Res. Bull. 1995; 36: 593-606Crossref PubMed Scopus (124) Google Scholar, 6Winkelmann J.C. Forget B.G. Blood. 1993; 81: 3137-3185Crossref Google Scholar, and 7Beck K.A. Nelson W.J. Am. J. Physiol. 1996; 270: C1263-C1270Crossref PubMed Google Scholar). Functional differences between the membranes of erythroid and nonerythroid cells argue against the simple erythrocyte model of the membrane skeleton. Major differences between the erythroid model and other cells include differences in the expression of spectrin (8Levine J. Willard M. J. Cell Biol. 1981; 90: 631-643Crossref PubMed Scopus (315) Google Scholar, 9Bennett V. Davis J. Fowler W. Nature. 1982; 299: 126-131Crossref PubMed Scopus (219) Google Scholar, 10Riederer B.M. Zagon I.S. Goodman S.R. J. Cell Biol. 1986; 102: 2088-2097Crossref PubMed Scopus (182) Google Scholar, 11Siman R. Ahdoot M. Lynch G. J. Neurosci. 1987; 7: 55-64Crossref PubMed Google Scholar) and ankyrin isoforms (12Kordeli E. Lambert S. Bennett V. J. Biol. Chem. 1994; 270: 2352-2359Abstract Full Text Full Text PDF Scopus (428) Google Scholar, 13Devarajan P. Stabach P.R. Mann A.S. Ardito T. Kashgarian M. Morrow J.S. J. Cell Biol. 1996; 133: 819-830Crossref PubMed Scopus (161) Google Scholar, 14Zhou D. Birkenmeier C.S. Williams M.W. Sharp J.J. Barker J.E. Bloch R.J. J. Cell Biol. 1997; 136: 621-631Crossref PubMed Scopus (89) Google Scholar, 15Gallagher P.G. Tse W.T. Scarpa A.L. Lux S.E. Forget B.G. J. Biol. Chem. 1997; 272: 19220-19228Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) (reviewed in Ref. 16Peter L.L. Lux S.E. Semin. Hematol. 1993; 30: 85-118PubMed Google Scholar), interactions of spectrin and ankyrin with additional proteins (17Sikorski A.F. Terlecki G. Zagon I.S. Goodman S.R. J. Cell Biol. 1991; 114: 313-318Crossref PubMed Scopus (87) Google Scholar, 18Harris A.S. Croall D.E. Morrow J.S. J. Biol. Chem. 1988; 263: 15754-15761Abstract Full Text PDF PubMed Google Scholar, 19Frappier T. Stetzkowski-Marden F. Pardel L.A. Biochem. J. 1991; 275: 521-527Crossref PubMed Scopus (37) Google Scholar, 20Riederer B.M. Goodman S.R. FEBS Lett. 1990; 277: 49-52Crossref PubMed Scopus (19) Google Scholar, 21Carlier M.F. Simon C. Gassoly R. Pradel G.A. Biochimie. 1984; 66: 305-311Crossref PubMed Scopus (47) Google Scholar), localization of spectrin in the cytoplasm as well as in the plasma membrane (10Riederer B.M. Zagon I.S. Goodman S.R. J. Cell Biol. 1986; 102: 2088-2097Crossref PubMed Scopus (182) Google Scholar, 11Siman R. Ahdoot M. Lynch G. J. Neurosci. 1987; 7: 55-64Crossref PubMed Google Scholar, 22Beck K.A. Buchanan J.A. Malhotra V. Nelson W.J. J. Cell Biol. 1994; 127: 707-723Crossref PubMed Scopus (168) Google Scholar), and the potential for dramatic rearrangements of spectrin's cellular location (23Jesaitis A.J. Bokoch G.M. Tolley J.O. Allen R.A. J. Cell Biol. 1988; 107: 921-928Crossref PubMed Scopus (61) Google Scholar, 24Perrin D. Langley O.K. Aunis D. Nature. 1987; 326: 498-501Crossref PubMed Scopus (136) Google Scholar) (reviewed in Refs. 2Morrow J.S. Rimm D.L. Kennedy S.P. Cianci C.D. Sinard J.H. Weed S.A. Hoffman J. Jamieson J. Handbook of Physiology. Oxford, London1996: 485-540Google Scholar and 7Beck K.A. Nelson W.J. Am. J. Physiol. 1996; 270: C1263-C1270Crossref PubMed Google Scholar). Several studies have demonstrated that both erythroid and nonerythroid spectrins are expressed in brain tissue (8Levine J. Willard M. J. Cell Biol. 1981; 90: 631-643Crossref PubMed Scopus (315) Google Scholar, 9Bennett V. Davis J. Fowler W. Nature. 1982; 299: 126-131Crossref PubMed Scopus (219) Google Scholar, 10Riederer B.M. Zagon I.S. Goodman S.R. J. Cell Biol. 1986; 102: 2088-2097Crossref PubMed Scopus (182) Google Scholar, 11Siman R. Ahdoot M. Lynch G. J. Neurosci. 1987; 7: 55-64Crossref PubMed Google Scholar, 25Clark M.B. Ma Y. Bloom M.L. Barker J.E. Zagon I.S. Zimmer W.E. Goodman S.R. Brain Res. 1993; 663: 223-236Crossref Scopus (35) Google Scholar). Neuronal compartmentalization of brain spectrin isoforms into axons and presynaptic terminals (nonerythroid spectrin) and into cell bodies and dendrites (erythroid spectrin) (10Riederer B.M. Zagon I.S. Goodman S.R. J. Cell Biol. 1986; 102: 2088-2097Crossref PubMed Scopus (182) Google Scholar, 25Clark M.B. Ma Y. Bloom M.L. Barker J.E. Zagon I.S. Zimmer W.E. Goodman S.R. Brain Res. 1993; 663: 223-236Crossref Scopus (35) Google Scholar) suggests that brain spectrin isoforms may perform related but distinct functions in neuronal cells. It has been suggested that nonerythroid spectrin performs a more general, constitutive role, while erythroid spectrin takes part in more specialized activities of differentiated cells (26Hu R.J. Watanabe M. Bennett V. J. Biol. Chem. 1992; 267: 18715-18722Abstract Full Text PDF PubMed Google Scholar). The α-subunit of erythroid spectrin, αI (27Sahr K.E. Laurila P. Kotula L. Scarpa A.L. Coupal E. Leto T.L. Linnenbach A.J. Winkelmann J.C. Speicher D.W. Marchesi V.T. Curtis P.J. Forget B.G. J. Biol. Chem. 1990; 265: 4434-4443Abstract Full Text PDF PubMed Google Scholar), 1Nomenclature for spectrin isoforms used in this paper is according to Winkelmann and Forget (6Winkelmann J.C. Forget B.G. Blood. 1993; 81: 3137-3185Crossref Google Scholar). and the α-subunit of nonerythroid spectrin, αII (28Wasenius V.M. Saraste M. Salven P. Eramaa M. Holm L. Lehto V.P. J. Cell Biol. 1989; 108: 79-93Crossref PubMed Scopus (124) Google Scholar, 29Moon R.T. McMahon A.P. J. Biol. Chem. 1990; 265: 4427-4433Abstract Full Text PDF PubMed Google Scholar), each contains a unique SH3 2The abbreviations used are: SH3, Src homology 3; GST, glutathione S-transferase; FISH, fluorescence in situ hybridization; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PCR, polymerase chain reaction; DIG, digoxygenin; PAC, P1 artificial chromosome; mAb, monoclonal antibody; BS, binding site; kb, kilobase pair(s); GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; TBS, Tris-buffered saline with Tween 20. domain. Distinct protein interactions are likely to involve these domains, and they may be important for specific distribution and specialized roles of brain spectrin isoforms. The SH3 domain was originally identified in the regulatory region of Src and Src-like tyrosine kinases (Abl, Fps) and then identified in other proteins, including Raf, phospholipase C, Ras GTPase-activating protein, and phosphatidylinositol 3′-kinase, all involved in transmitting signals within cells (reviewed in Refs. 30Koch A. Anderson D. Morgan M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1444) Google Scholar, 31Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 32Birge B.R. Knudsen B.S. Besser D. Hanafusa H. Genes Cells. 1996; 1: 595-613Crossref PubMed Scopus (122) Google Scholar). The SH3 domains are involved in protein interactions thought to control signaling pathways. In Src and Abl, oncogenic mutations have been identified in the SH3 domain, indicating that this region might have a negative regulatory effect on transformation (33Hirai H. Varmus H.E. Mol. Cell. Biol. 1990; 10: 1307-1318Crossref PubMed Google Scholar). The SH3 domains of several tyrosine kinases were found to bind to short proline-rich sequences containing a PXXP motif (34Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar, 35Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar) and a general model for the SH3-ligand complex has been proposed based on NMR studies (36Yu H. Rosen M. Shin T.B. Seidel-Dugan C. Brugge J.S. Schreiber S.L. Science. 1992; 258: 1665-1669Crossref PubMed Scopus (285) Google Scholar, 37Feng S. Chen J.K. Yu H. Simon J.A. Schreiber S.L. Science. 1994; 266: 1241-1247Crossref PubMed Scopus (747) Google Scholar). The list of SH3-containing and SH3-binding proteins is rapidly growing (reviewed in Ref. 32Birge B.R. Knudsen B.S. Besser D. Hanafusa H. Genes Cells. 1996; 1: 595-613Crossref PubMed Scopus (122) Google Scholar). Diversity in SH3 domains and in their ligand binding sites indicate that their binding specificities are variable and thus mediate different protein-protein interactions. In addition to αI and αII spectrin, several unrelated cytoskeletal or membrane proteins have been shown to contain the SH3 domain, including a major palmitoylated erythrocyte membrane protein p55 (38Ruff P. Speicher D.W. Husain-Chishti A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6595-6599Crossref PubMed Scopus (139) Google Scholar), several isoforms of myosin Ib, a yeast actin-binding protein implicated in the regulation of cytoskeletal assembly (39Drubin D.G. Mulholland J. Zhu Z. Botstein D. Nature. 1990; 343: 288-290Crossref PubMed Scopus (205) Google Scholar), and yeast protein BEM1, which is involved in cell polarization (40Chenevert J. Corrado K. Bender A. Pringle J. Herskowitz I. Nature. 1992; 356: 77-79Crossref PubMed Scopus (158) Google Scholar). Using interaction cloning, we identified a cDNA encoding a candidate human αI spectrin SH3 domain-binding protein. Five isoforms of the candidate mRNA were identified in human brain. Using the recombinant polypeptides, we located the αI SH3 domain and Abl-SH3 domain binding regions. Immunofluorescence studies suggest association of the αI spectrin SH3 domain-binding protein and Abl tyrosine kinase with an erythroid-like spectrin in transfected NIH3T3 cells. The candidate αI SH3-binding protein belongs to a recently identified family of tyrosine kinase-binding proteins (41Shi Y. Alin K. Goff S. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar, 42Zonghan D. Pendegrast M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 43Wang B. Mysliwiec T. Krainc D. Jensen R.A. Sonoda G. Testa J.R. Golemis E.A. Kruh G.D. Oncogene. 1996; 12: 1921-1929PubMed Google Scholar, 44Biesova Z. Piccoli C. Wong W.T. Oncogene. 1997; 14: 233-241Crossref PubMed Scopus (90) Google Scholar), and one of its isoforms is identical to e3B1, an eps8-binding protein (44Biesova Z. Piccoli C. Wong W.T. Oncogene. 1997; 14: 233-241Crossref PubMed Scopus (90) Google Scholar). Based on conservation of the spectrin SH3 binding site within members of this protein family and published data (41Shi Y. Alin K. Goff S. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar, 42Zonghan D. Pendegrast M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 43Wang B. Mysliwiec T. Krainc D. Jensen R.A. Sonoda G. Testa J.R. Golemis E.A. Kruh G.D. Oncogene. 1996; 12: 1921-1929PubMed Google Scholar, 44Biesova Z. Piccoli C. Wong W.T. Oncogene. 1997; 14: 233-241Crossref PubMed Scopus (90) Google Scholar), a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed. The sequence encoding the αI spectrin SH3 domain (nucleotides 3115–3291 of the αSpI cDNA; Ref. 27Sahr K.E. Laurila P. Kotula L. Scarpa A.L. Coupal E. Leto T.L. Linnenbach A.J. Winkelmann J.C. Speicher D.W. Marchesi V.T. Curtis P.J. Forget B.G. J. Biol. Chem. 1990; 265: 4434-4443Abstract Full Text PDF PubMed Google Scholar) and the αII spectrin SH3 domain (nucleotides 2995–3177 of the αSpII cDNA; Ref. 29Moon R.T. McMahon A.P. J. Biol. Chem. 1990; 265: 4427-4433Abstract Full Text PDF PubMed Google Scholar) were amplified using specific primers (see Table I) and subcloned into the pAS vector; the αI SH3 domain plasmid is called pAS-Sp, and the αII SH3 domain plasmid is called pAS-F. In both cases, the SH3 domain sequence was fused to the C terminus of the GAL4 DNA binding domain. The human adult brain expression library was obtained from CLONTECH(catalog no. HL4004AB), and the library screening was performed using the yeast strain Y190, with addition of 25 mm aminotriazole into the medium. Yeast transformations and library screening were performed as described in Ref. 45Fields S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar. For the liquid β-galactosidase assays, chlorophenol red-β-d-galactopyranoside was used as the substrate.Table IOligonucleotide primers for the expression plasmidsPlasmidPrimer namePrimer sequencepAS-SpYSSH5′5′-GAGAATTCCATGGCTGGAGAACAAAGGGTC-3′YSSH3′5′-GAAGGATCCTCAGGCCAGTCTTCTGAC-3′pAS-FYFSH5′5′-GCGAATTCCATGGATGAGACTGGGAAGG-3′YFSH3′5′-TGAGGATCCTCAGTCCAATTTCTTCACG-3′GST-E-SH3SH5′15′-GAGGGATCCGCTGGAGAACAAAGGGTC-3′SH3′15′-GCGGAATTCGTTCTCAATCTGCTCCTG-3′GST-F-SH3NSH35′15′-CCCGGATCCGATGAGACTGGGAAGG-3′NSH33′15′-TGAGAATTCTCAGTCCAATTTCTTCACG-3′N3–1M5′5′-GGGGGATCCATGGCAGAGCTGCAGATGTTAC-3′NGFP413′5′-GCGAATTCATCAGTATAGTGCATGATTGATTCAAC-3′C1–2E-1SH5′15′-GAGGGATCCGCTGGAGAACAAAGGGTC-3′2ESH3′5′-AGGGAATTCATTGGTTCTCAATCTGCTCCTGG-3′C4T15′5′-ATCGGATCCACAGTTCTGGATGATG-3′E3′5′-ACTGAATTCAAGACTCCTTCATACC-3′C5T25′5′-GTTAGATCTAATGATCCATATGCAGATGGG-3′E3′See aboveC6 and C7T15′See aboveT33′5′-GCAAGATCTCCATCTGCATATGGATCATTATAC-3′C8BS35′5′-AGAGGATCCCCTCTTACTCAGAAAC-3′T33′See aboveC9BS45′5′-AGTGGATCCAGTGGAGGAAGTGGAAGTCG-3′T33′See aboveC10BS15′5′-TTGGATCCCCCATTGCTGTGCCTACAC-3′T33′See aboveC11WS15′5′-ATTGGATCCGTTTCTATTGCTCCACCCCCTCCCCCTATGCCTCAGTTGACTCCACAGATACCTCTCACA-3′T33′See aboveC13BS65′5′-GAAGGATCCGCTGATAGTCCAACTCCACCGCCAC-3′T33′See aboveC15WS15′See aboveWS23′5′-GGAGAATTCTAAATGTTTTCCTGCACCCTGGCCACGAAGCCTGTGAGAGGTATCTGTGG-3′C16T15′5′-ATCGGATCCACAGTTCTGGATGATG-3′WS23′See aboveC17T15′See aboveBS23′5′-CTGAATTCACAACTGAGGCATAGGG-3′C18T15′See aboveBS73′5′-GCAGAATTCTATGGTCCAATAGTGGGTGGC-3′C19T15′See aboveG3′5′-TGGGAATTCAATACTACTGCTACCAC-3′ Open table in a new tab Marathon-Ready human brain cDNA and Klentaq DNA polymerase mix (CLONTECH) were used to clone the 5′ (primer A3′: 5′-TATGAATTCGCTGGAGTACATTGTTG-3′) and 3′ (primer T25′, Table I) ends of the hssh3bp1/e3B1 mRNA and to clone its isoforms. Although no additional sequence was obtained at the 5′ end, the 3′-untranslated region was extended to 1044 base pairs; it contained a poly(A) addition signal, AATAAA, located 17 base pairs from the 3′ poly(A) tail. At the 5′ end of the hssh3bp1/e3B1 cDNA, the first ATG codon was located 81 base pairs downstream from the 5′EcoRI restriction site. No additional ATG codons were found following the TATA box identified in the genomic DNA fragment that hybridized to the 5′ end sequence (data not shown); therefore, it is likely that the first ATG identified in the original cDNA clone represents the translation initiation site of the candidate mRNA. The amplified DNA fragments were cloned into M13mp18 or 19 or into pGEX2T vector (Amersham Pharmacia Biotech) and sequenced (46Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Each hssh3bp1/e3B1 isoform was sequenced from two independent subclones. The variable alanine residue (see Fig. 1 A) was present in five of eight subclones. The desired regions of αI and αII spectrin were obtained by amplification using specific primers and subsequently cloned into the pGEX-2T vector using BamHI andEcoRI restriction sites incorporated into amplification products by primers. All primer sequences used for expression plasmids are listed in Table I. The plasmid GST-E-SH3 encodes residues 977–1062 of the αI spectrin that includes the SH3 domain (residues 3115–3370 in the αI cDNA; Ref. 27Sahr K.E. Laurila P. Kotula L. Scarpa A.L. Coupal E. Leto T.L. Linnenbach A.J. Winkelmann J.C. Speicher D.W. Marchesi V.T. Curtis P.J. Forget B.G. J. Biol. Chem. 1990; 265: 4434-4443Abstract Full Text PDF PubMed Google Scholar). In this case, the termination codon was present in the vector sequences, and this resulted in addition of residues EFIVTD to the C terminus of the spectrin sequence. The construct GST-F-SH3 encodes residues 965–1025 of αII spectrin (nucleotides 2995–3177 of αSpII cDNA sequence; Ref. 29Moon R.T. McMahon A.P. J. Biol. Chem. 1990; 265: 4427-4433Abstract Full Text PDF PubMed Google Scholar). The nucleotide sequences of all plasmid constructs were confirmed by DNA sequencing. pGEX-2T plasmids expressing SH3 domains of Abl, Crk, Src and n-Src (34Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar, 35Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar) were obtained from Dr. Bruce J. Mayer (Howard Hughes Medical Institute, Children's Hospital, Boston, MA). The 1.63-kb EcoRI fragment was obtained from the clone pGAD4-1 (isolated from the human brain expression library) and subcloned into the plasmid pGEX1λ (Amersham Pharmacia Biotech), plasmid C1. The other GST-hssh3bp1/e3B1 fusion plasmids are pGEX-2T subclones. Plasmids C2 and C3 were obtained by truncation of the 1.63-kb fragment at the BamHI site andNcoI site, respectively. The plasmid C12 was obtained by subcloning the HincII fragment of the 1.63-kbEcoRI fragment, clone pGAD4-1, into the SmaI site of pGEX-2T. All subsequent plasmids are subclones of amplification products obtained using specific primers listed in Table I and the clone pGAD4-1 as a template; for the plasmid C7, the PCR fragment encoding isoform 5 was used. The plasmid C4 was obtained by subcloning the amplification product digested with BamHI. The plasmid C5 was obtained by ligating the BglII-BamHI fragment obtained from the PCR amplification with theBamHI-EcoRI fragment from the plasmid C12 into the BamHI-EcoRI sites of pGEX2T and checking for the correct orientation of inserts. Plasmids C6, C8–C10, and C13 were assembled in each case by subcloning simultaneously theBamHI-BglII fragment obtained from the PCR amplification and the BamHI-EcoRI fragment from the plasmid C12 as described for C5. The plasmid C15 was assembled by annealing two complementary primers. The rest of the plasmids (C16–C19) are PCR amplification products subcloned into theBamHI and EcoRI restriction sites of pGEX-2T. The coding sequence of hssh3bp1/e3B1 isoform 1 was obtained by PCR amplification (primers M5′ and NGFP413′) and was subcloned into the plasmid pEGFP-N3 (BglII and EcoRI sites) (CLONTECH) so that the GFP sequences were located at the C terminus of hssh3bp1/e3B1 (plasmid N3–1). The αI SH3 domain containing the same region of αI spectrin as clone GST-E-SH3 was obtained by PCR amplification (primers SH5–1 and 2E-SH3) and cloned into the BglII and EcoRI sites of the plasmid pEGFP-C1 (CLONTECH) (plasmid C1–2E1). In plasmid NG-1, the GFP sequence was removed from plasmid N3–1 by digestion withEcoRI and BsrGI restriction enzymes followed by the Klenow fill-in reaction and ligation. The affinity purification of GST fusion proteins on glutathione-Sepharose was followed by gel filtration using Sephacryl S-100 (47Kotula L. DeSilva T.M. Speicher D.W. Curtis P.J. J. Biol. Chem. 1993; 268: 14788-14793Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were estimated using the bicinchoninic acid assay (Pierce). Biotinylation of recombinant polypeptides was performed using 6-[(6-{(biotinoyl)amino}hexanoyl)amino]hexanoic acid, succinimidyl ester (Biotin-XX, SE; Molecular Probes, Eugene, OR), and the filter binding assay was performed essentially as described (34Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar,35Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar). All GST fusion proteins were expressed in BL21 strain ofEscherichia coli. Inductions of GST recombinant polypeptides were performed as described (47Kotula L. DeSilva T.M. Speicher D.W. Curtis P.J. J. Biol. Chem. 1993; 268: 14788-14793Abstract Full Text PDF PubMed Google Scholar). In each case, bacterial lysates from 50 μl of induced cell cultures were solubilized and separated on SDS-Tricine polyacrylamide gels and blotted onto polyvinylidene difluoride membranes. After the transfer, blots were blocked in TBST buffer (20 mm Tris-HCl, 150 mm NaCl, 0.05% Tween 20, v/v) containing 2% nonfat dried milk for 1 h at room temperature. The blots were then incubated in the same buffer with biotinylated recombinant fusion proteins (0.4 μg/ml). Following incubation for 1 h at 20 °C, the blots were extensively washed with TBST buffer and incubated with a streptavidin-alkaline phosphatase conjugate at 1:5,000 dilution (Boehringer Mannheim) in 2% milk TBST for 1 h. After washing, the blots were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates. The band located between the 19.8- and 33.5-kDa molecular mass markers, present on all blots developed with the streptavidin-alkaline phosphatase conjugate, most likely represents a naturally occurring biotinylated bacterial protein and has been observed previously (34Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar, 35Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar). To quantitate the intensity of bands, the blots were scanned and bands were quantified using Scan Analysis software (Biosoft, Ferguson, MO). NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with glutamine, 10% bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were transiently transfected with GFP fusion plasmids or NG-1 using LipofectAMINE (Life Technologies, Inc.). 24–48 h after transfection, cells were fixed with −20 °C methanol and 1 mm EGTA, and processed for imunofluorescence essentially as described in Ref. 63Holleran E.A. Tokito M.K. Karki S. Holzbaur E.L.F. J. Cell Biol. 1996; 135: 1815-1829Crossref PubMed Scopus (197) Google Scholar. Texas red-conjugated goat anti-rabbit secondary antibody (Molecular Probes) was used in all experiments; no staining of GFP alone was observed with this antibody. All images were obtained from the Texas Red channel first, and this procedure prevented possible bleed-through of GFP immunofluorescence into the Texas Red channel. For experiments with plasmid NG-1, hssh3bp1/e3B1 was stained with mAb 4E2 raised to GST-hssh3bp1/e3B1, 3L. Kotula and K. S. Kim, unpublished data. followed by FITC-conjugated goat anti-mouse secondary antibody (Molecular Probes). The polyclonal antibody 992 (anti-axonal brain spectrin 240/325 or anti-αII/βII) was purchased from Chemicon International Inc., Temecula, CA (10Riederer B.M. Zagon I.S. Goodman S.R. J. Cell Biol. 1986; 102: 2088-2097Crossref PubMed Scopus (182) Google Scholar). The polyclonal antibody raised against human red cell spectrin αIΣI/βIΣI (anti-HS) was obtained from ICN Biomedicals, Costa Mesa, CA. The polyclonal antibody raised against canine erythrocyte spectrin, which recognizes Golgi spectrin βIΣ*, was a generous gift from Drs. Kenneth Beck and James Nelson (Stanford University, Stanford, CA) (22Beck K.A. Buchanan J.A. Malhotra V. Nelson W.J. J. Cell Biol. 1994; 127: 707-723Crossref PubMed Scopus (168) Google Scholar). The polyclonal antibody raised against the 80-kDa domain of αIΣI erythrocyte spectrin was a generous gift from Dr. David W. Speicher (The Wistar Institute, Philadelphia, PA). The polyclonal antibody against βI spectrin was obtained from Affinity Bioreagents, Golden, CO (59Porter G.A. Scher M.G. Resneck W.G. Porter N.C. Fowler V.M. Bloch R.J. Cell Motil. Cytoskeleton. 1997; 37: 7-19Crossref PubMed Scopus (23) Google Scholar). The polyclonal antibody against Abl tyrosine kinase (Ab1) was obtained from Calbiochem-Novabiochem International, La Jolla, CA. The monoclonal antibody against GST was raised at the IBR Antibody Facility using standard techniques (49Kim K.S. Miller D.L. Sapienza V.J. Chun Bai, C. Grundke-Iqbal I. Currie J.R. Wisniewski H.M. Neurosci. Res. Commun. 1988; 2: 121-130Google Scholar). A human P1 artificial chromosome (PAC) (50Ioannou P. Amemiya C. Garnes J. Kroisel P. Shizuya H. Chen C. Batzer M. de Jong P. Nat. Genet. 1994; 6: 84-89Crossref PubMed Scopus (764) Google Scholar) l" @default.
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