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) mere produced v ia the baculovirus expression vector system, Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell l ine (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C Delta 795)), or an amino-terminal deletion m utant containing the putative catalytic domain ((N Delta 661)rJAK2). T he proteins produced in infected Sf21 cells were assayed for phosphoty rosine content and autophosphorylating activity. Tyrosine phosphorylat ion of rJAK2 was not observed 1 day postinfection when rJAK2 was initi ally produced but was apparent 2 or more days postinfection when the r JAK2 level had significantly increased. Tyrosine phosphorylation of rJ AK2(C Delta 795) was not observed; further, coproduction of rJAK2(C De lta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consist ent with previously published results (Zhuang, H., Patel, S. V., He, T -C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. C hem. 269, 21411-21414). Mutant (N Gamma 661)rJAK2 exhibited a robust t yrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotei n co-immunoprecipitated with (N Delta 661)rJAK2 but not with rJAK2 or rJAK2(C Delta 795). Both rJAK2 and (N Delta 661)rJAK2 incorporated pho sphate under in vitro kinase assay conditions, but rJAK2(C Delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhib itory sites would provide a simple model to describe these results.