IDENTIFICATION OF A CANDIDATE HUMAN SPECTRIN SRC HOMOLOGY-3 DOMAIN-BINDING PROTEIN SUGGESTS A GENERAL MECHANISM OF ASSOCIATION OF TYROSINE KINASES WITH THE SPECTRIN-BASED MEMBRANE SKELETON

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 spectri n SH3 domain-binding protein was identified by interaction screening o f a human brain expression library using the human erythroid spectrin (alpha I) SH3 domain as a bait. Five isoforms of the alpha I SH3 domai n-binding protein mRNA were identified in human brain, Mapping of SH3 binding regions revealed the presence of two alpha I SH3 domain bindin g regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to hum an chromosome 10p11.2 --> p12. The gene belongs to a recently identifi ed 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 characteri stic of the reticulovesicular system. This fluorescence pattern was si milar to that obtained with the anti-human erythroid spectrin alpha I Sigma I/beta I Sigma I antibody in untransfected NIH3T3 cells; in addi tion, the anti-alpha I Sigma I/beta I Sigma I antibody also stained Go lgi apparatus. Immunofluorescence obtained using antibodies against al pha I Sigma I/beta I Sigma I spectrin and Abl tyrosine kinase but not against alpha II/beta II spectrin colocalized with the overexpressed g reen fluorescent protein-SH3-binding protein. Based on the conservatio n of the spectrin SH3 binding site within members of this protein fami ly and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed .