A strong protein-tyrosine kinase activity is associated with a baculovirus-expressed chicken tkl gene.
We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein-tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte-specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51-kDa Tkl protein (p51tkl) at 40 h post-infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein-tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.
Gärtner
T
T
Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt, Federal Republic of Germany.
Kühnel
H
H
Raab
G
G
Raab
M
M
Strebhardt
K
K
Rübsamen-Waigmann
H
H
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D003062:Codon; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D005796:Genes; D016391:Genes, src; D007313:Insecta; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000201:enzymology; D014162:Transfection
D000596:Amino Acids; D003062:Codon; D011994:Recombinant Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases