Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis.
By using antibodies raised against a portion of N terminus of 40-kDa kinase (Kobayashi, T., Nakamura, S., Taniguchi, T., and Yamamura, H. (1990) Eur. J. Biochem. 188, 535-540), not only 40-kDa protein but also 72-kDa protein were detected on immunoblot analysis of porcine spleen homogenate. In splenocytes preparation, the antibodies could immunoprecipitate protein-tyrosine kinase activity of the 72-kDa protein but not detect the 40-kDa protein even on immunoblot. After incubation of crude spleen homogenate at 37 degrees C with or without various protease inhibitors, immunoblot analysis revealed proteolytic breakdown of the 72-kDa protein to 40-kDa fragment. Next, using oligonucleotides designed according to partially sequenced information of the 40-kDa kinase as a probe, we have isolated a clone containing entire coding sequence for the 40-kDa kinase from a porcine spleen cDNA library. This clone had a 1884-base-pair-long open reading frame encoding 628-amino-acid polypeptide with a calculated molecular weight of 71,618. The deduced amino acid sequence did not contain a ligand binding or membrane spanning region but did a well-conserved protein-tyrosine kinase domain and two src homology region 2 domains. The sequences of these domains showed 30-40% identity to those of other protein-tyrosine kinases, but those of remaining parts were quite unique. From these results, we concluded that the 40-kDa kinase was generated by proteolysis from the 72-kDa holoprotein which was a new member of nonreceptor protein-tyrosine kinase so far reported. We therefore designated this gene as syk after spleen tyrosine kinase.
Taniguchi
T
T
Department of Biochemistry, Fukui Medical School, Japan.
Kobayashi
T
T
Kondo
J
J
Takahashi
K
K
Nakamura
H
H
Suzuki
J
J
Nagai
K
K
Yamada
T
T
Nakamura
S
S
Yamamura
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000201:enzymology; D013552:Swine
D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases