Prolactin induces Jak2 phosphorylation of RUSHY195.

Jak2/RUSH-mediated prolactin signaling culminates in RUSH-1α-DNA-binding. Heretofore, Jak2-specific phosphorylation residues in RUSH were unknown. Genpathway's discovery approaches correlated RUSH-DNA binding (-126/-121) in uteroglobin's proximal promoter with recruitment of the transcriptional machinery. NetPhos 2.0 server found a single tyrosine phosphorylation site in RUSH's minimal DNA-binding domain. Y195 had identical context and prediction scores (0.52) for rabbit and human (HLTF) orthologs. The mouse ortholog (Hltf) had a higher prediction score (0.897). Affinity purified RUSHY195ph antibodies recognized native tyrosine phosphorylated RUSH protein immunoprecipitated from nuclear extracts. When R5020-treated HRE-H9 cells±the Jak2 inhibitor, Tyrene CR4, were stimulated with prolactin, confocal immunofluorescence images provided conclusive evidence that Jak2 mediated the availability of phosphorylated RUSHY195 in nucleus and cytoplasm. Catalytically active Jak2 is ipso facto a RUSH site-specific tyrosine kinase. Immunoprecipitation/Western blotting revealed both phosphorylation at Y195 and the physical interaction between p-Jak2/RUSH/HLTF/Hltf are evolutionarily conserved across three mammalian (rabbit, human, mouse) orthologs.