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

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 Ab1-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, 233241). 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 Ab1 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.