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 whi ch is suggested to be expressed in hematopoietic cells and neuronal ce lls in human. Recently we have cloned a full sized human ltk cDNA whic h has a 423 amino acid extracellular domain which may bind to unknown ligand(s), and a 415 amino acid cytoplasmic domain which contains a ty rosine kinase domain. To identify the cellular signal transducer prote ins binding to the ltk protein, we have analysed the recombinant ltk p rotein transiently expressed in COS cells. By an in vitro immune compl ex kinase assay, a major 140 kDa phosphoprotein and other cellular pho sphoproteins were co-immunoprecipitated with the 100 kDa ltk protein u sing anti-ltk monoclonal antibodies. Western blot analysis revealed th at the wild-type ltk protein was tyrosine-phosphorylated in vivo and a ssociated with SH2 containing proteins, PLC-gamma 1, p85 subunit of PI 3-K and GAP, in vivo. Furthermore, the wild-type ltk protein also bind s to a serine/threonine kinase, Raf-1, in vivo. In contrast, none of t hese signal transducer proteins were associated with a kinase-negative ltk mutant (K544M-ltk) in which methionine at the putative ATP bindin g site was replaced with lysine. These results suggest that the associ ations of the ltk protein with those signaling molecules depend on the tyrosine kinase activity of the ltk protein. This is the first detect ion of cytoplasmic signal transducers that bind to the ltk protein in vivo.