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 (alphaI) SH3 domain as a bait. Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two alphaI 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)Oncogene 14, 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 alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII 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.
Highlights
Erythroid spectrin is the predominant component of the twodimensional protein network called the membrane skeleton, underlying the lipid bilayer of red cells
Yeast Two-hybrid Screening for Spectrin Src homology 3 (SH3) Domain-binding Proteins—The yeast two-hybrid system was used to screen the human brain cDNA expression library for spectrin SH3 domain-binding proteins
Clone pGAD4-1 showed over 33fold above background -galactosidase activity when transformed with the construct containing the ␣I spectrin SH3 domain, indicating an interaction
Summary
Expression of the ␣I and ␣II SH3 Domain in Yeast, and Expression Library Screening—The sequence encoding the ␣I spectrin SH3 domain (nucleotides 3115–3291 of the ␣SpI cDNA; Ref. 27) and the ␣II spectrin SH3 domain (nucleotides 2995–3177 of the ␣SpII cDNA; Ref. 29) were amplified using specific primers (see Table I) and subcloned into the pAS vector; the ␣I SH3 domain plasmid is called pAS-Sp, and the ␣II SH3 domain plasmid is called pAS-F. 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. Green Fluorescent Protein (GFP) Fusions—The coding sequence of hssh3bp1/e3B1 isoform 1 was obtained by PCR amplification (primers M5Ј and NGFP413Ј) 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 ␣I SH3 domain containing the same region of ␣I 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). The blots were developed with nitro blue tetrazolium/5-bromo-
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