Activation and functional analysis of Janus kinase 2 in BA/F3 cells using the coumermycin/gyrase B system.
Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3- or granulocyte-macrophage colony-stimulating factor-mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)-Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB-Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB-Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.
Mohi
M G
MG
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
Arai
K i
Ki
Watanabe
S
S
D049933:Aminocoumarins; D000818:Animals; D019556:COS Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D053614:Janus Kinase 2; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D049933:Aminocoumarins; D003374:Coumarins; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C004628:coumermycin