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

doi:10.1074/jbc.273.22.13681

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*

From the New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314

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)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 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.

Footnotes

↵* This work was supported by National Institutes of Health NINDS Grant R29 NS32874 (to L. K.) and the New York State Office of Mental Retardation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U87166.
↵‡ To whom correspondence should be addressed: Laboratory of Molecular Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5160; Fax: 718-698-3803; E-mail:kotulal{at}interport.net.
↵1 Nomenclature for spectrin isoforms used in this paper is according to Winkelmann and Forget (6).
↵2 The 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.
↵3 L. Kotula and K. S. Kim, unpublished data.
↵4 Gene symbols SSH3BP1 andSSH3BP2 have been approved by the Human Genome Organization (HUGO) Nomenclature Committee, London, United Kingdom.
↵5 L. Kotula, unpublished data.
- Received October 23, 1997.
- Revision received January 30, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.