Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.
Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.
He
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School of Pharmacy, University of Maryland, 20 North Pine Street, Baltimore, MD 21201, USA.
Li
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Schembri-King
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Jakes
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Hayashi
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D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003285:Contractile Proteins / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008840:Microfilament Proteins / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003285:Contractile Proteins; D064448:Filamins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D011506:Proteins; C490778:SH2B3 protein, human