Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth.
Eps8, a substrate of receptor tyrosine kinases, is an SH3 domain containing protein that plays an important role in mitogenic signaling. To determine the cellular function of eps8, we used the SH3 domain of eps8 to screen a human fibroblast M426 expression library and identified, a full-length cDNA clone of 3.2 kb. We designated this clone e3B1 for eps8 SH3 domain binding protein 1. Northern analysis revealed that expression of e3B1 mRNA was ubiquitous in human tissues. The e3B1 gene encodes a SH3 domain containing protein. We show that anti-e3B1 antibodies detect three cytosolic protein species of 65, 68 and 72 kDa in cell lysate isolated from asynchronously growing NIH3T3 cells. E3B1 binds to the SH3 domain of eps8 and Abl in vitro. We also demonstrated that e3B1 associates with eps8 in vivo. Phosphatase digestion and phosphoamino acid analysis revealed that p65e3B1 is a phosphoserine containing protein and p72e3B1 and p68e3B1 are hyperserine-phosphorylated form of p65e3B1. We further determined that the p65e3B1 was the most abundant in serum-starved NIH/EGFR cells. Time course studies initiated by the addition of epidermal growth factor (EGF) revealed that the p72e3B1 started to accumulate at 4 h, peaked at 8 h and remained high until 24 h. Finally, we demonstrate that NIH/EGFR fibroblasts overexpressing e3B1 grow more slowly relative to matched controls.
Biesova
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Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Piccoli
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Wong
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D016475:3T3 Cells / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D018909:src Homology Domains / Q000235:genetics*
C513733:ABI1 protein, human; C513734:Abi1 protein, mouse; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D004815:Epidermal Growth Factor