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 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.
Ziemnicka-Kotula
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D
New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
Xu
J
J
Gu
H
H
Potempska
A
A
Kim
K S
KS
Jenkins
E C
EC
Trenkner
E
E
Kotula
L
L
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018909:src Homology Domains
C513733:ABI1 protein, human; C513734:Abi1 protein, mouse; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013049:Spectrin; D011505:Protein-Tyrosine Kinases