Human ltk receptor tyrosine kinase binds to PLC-gamma 1, PI3-K, GAP and Raf-1 in vivo.
Leukocyte tyrosine kinase (ltk) is a receptor-type tyrosine kinase which is suggested to be expressed in hematopoietic cells and neuronal cells in human. Recently we have cloned a full sized human ltk cDNA which has a 423 amino acid extracellular domain which may bind to unknown ligand(s), and a 415 amino acid cytoplasmic domain which contains a tyrosine kinase domain. To identify the cellular signal transducer proteins binding to the ltk protein, we have analysed the recombinant ltk protein transiently expressed in COS cells. By an in vitro immune complex kinase assay, a major 140 kDa phosphoprotein and other cellular phosphoproteins were co-immunoprecipitated with the 100 kDa ltk protein using anti-ltk monoclonal antibodies. Western blot analysis revealed that the wild-type ltk protein was tyrosine-phosphorylated in vivo and associated with SH2 containing proteins, PLC-gamma 1, p85 subunit of PI3-K and GAP, in vivo. Furthermore, the wild-type ltk protein also binds to a serine/threonine kinase, Raf-1, in vivo. In contrast, none of these signal transducer proteins were associated with a kinase-negative ltk mutant (K544M-ltk) in which methionine at the putative ATP binding site was replaced with lysine. These results suggest that the associations of the ltk protein with those signaling molecules depend on the tyrosine kinase activity of the ltk protein. This is the first detection of cytoplasmic signal transducers that bind to the ltk protein in vivo.
Kozutsumi
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Toyoshima
H
H
Hagiwara
K
K
Yazaki
Y
Y
Hirai
H
H
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D020794:Receptor Protein-Tyrosine Kinases; D011971:Receptors, Immunologic; D010738:Type C Phospholipases; D014443:Tyrosine; C489300:LTK protein, human