Characterization of active and inactive forms of the JAK2 protein-tyrosine kinase produced via the baculovirus expression vector system.
Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results.
Duhé
R J
RJ
Biological Carcinogenesis and Development Program, Science Applications International Corporation, Frederick, Maryland 21702-1201, USA.
Farrar
W L
WL
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D003001:Cloning, Molecular; D053614:Janus Kinase 2; D007700:Kinetics; D016296:Mutagenesis; D019000:Phosphotyrosine / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D018411:Spodoptera; D013997:Time Factors; D014162:Transfection
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2