Characterization of the tyrosine kinase Tnk1 and its binding with phospholipase C-gamma1.
Tnk1 is a nonreceptor tyrosine kinase cloned from CD34+/Lin-/CD38- hematopoietic stem/progenitor cells. The cDNA predicts a 72-kDa protein containing an NH(2)-terminal kinase, a Src Homology 3 (SH3) domain, and a proline-rich (PR) tail. We generated rabbit antiserum to a GST-Tnk1(SH3) fusion protein. Affinity-purified anti-Tnk1 antibodies specifically recognized a 72-kDa protein in Tnk1-transfected COS-1 cells and cells which express Tnk1 mRNA. Western blot analysis indicated that Tnk1 is expressed in fetal blood cells, but not in any other hematopoietic tissues examined. Tnk1 immunoprecipitated from cell lysates possessed kinase activity and was tyrosine phosphorylated. In binding experiments with a panel of GST-fusion constructs, only GST-PLC-gamma1(SH3) interacted with in vitro translated Tnk1. GST-protein precipitations from cell lysates confirmed that GST-PLC-gamma1(SH3) associated with endogenously expressed Tnk1. Conversely, GST-Tnk1(PR) protein constructs complexed with endogenously expressed PLC-gamma1. The association of Tnk1 with PLC-gamma1 suggests a role for Tnk1 in phospholipid signal transduction.
Felschow
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Johns Hopkins Oncology Center, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, Maryland 21231, USA.
Civin
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Hoehn
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D005326:Fetal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
D000906:Antibodies; D005326:Fetal Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C098672:TNK1 protein, human; D010738:Type C Phospholipases; D051966:Phospholipase C gamma