JAK2-STAT5 INTERACTIONS ANALYZED IN YEAST

Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) a nd signal transducers and activators of transcription (Stats) for nucl ear signaling, Here, we have established yeast strains in which an aut oactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed S tat5 protein. Transcriptional activity of Stat5 on a stably integrated , Stat-dependent reporter gene required the C-terminal fusion of the V P16 transactivation domain. In such yeast strains, the interaction bet ween Jak2 and Stat5 was analyzed without interference by other mammali an proteins involved in regulating Jak-Stat signaling, and mutant vers ions of both proteins were analyzed for their ability to productively interact. Complexes between Jab2 and Stat5 were found to be stable und er stringent co-immunoprecipitation conditions. Deletion of the Jak ho mology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain ( JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic acti vity, A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain intera ction was sufficient for the dimerization of Stat5, but such dimers bo und to DNA very inefficiently. Together, our data show that yeast cell s are appropriate tools for studying Jak-Stat or Stat-Stat interaction s. Our mutational analysis suggests that the Stat5 SH2 domain is essen tial for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase do main may be all that is needed to cause Stat phosphorylation in situat ions where receptor docking is dispensable.