Activation of receptor-associated tyrosine kinase JAK2 by prolactin.
JAK family tyrosine kinases have recently been implicated in intracellular signal transduction by transmembrane cytokine receptors of the interferon (IFN) and hematopoietin receptor families. Using the prolactin (PRL)-dependent rat pre-T cell line Nb2, a PRL receptor-associated, candidate tyrosine kinase of 120-130 kDa was recently characterized (1). In the present work this protein is identified as JAK2, based upon reciprocal anti-JAK2 and anti-phosphotyrosine immunoprecipitation and immunoblotting. JAK2 underwent rapid and transient tyrosine phosphorylation in response to receptor activation, reaching peak levels within 5 min of exposure to 100 nM PRL at 37 degrees C. In vitro tyrosine kinase assays using either [gamma-32P]ATP and autoradiography or unlabeled ATP combined with anti-phosphotyrosine immunoblotting, demonstrated that the activity of JAK2 was stimulated by PRL. Phosphoamino acid analysis of JAK2 after in vitro tyrosine kinase assay revealed that the majority of phosphate was incorporated into tyrosine residues. Furthermore, JAK2 was associated with PRL receptors to a comparable extent before and after PRL binding, as demonstrated by anti-receptor immunoprecipitation and subsequent anti-JAK2 immunoblotting. We propose that binding of ligand to the PRL receptor activates preassociated JAK2, and that this enzyme generates the initial signal in the intracellular communication cascade.
Rui
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Cytokine Molecular Mechanisms Section, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702.
Kirken
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Farrar
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D004789:Enzyme Activation; D015151:Immunoblotting; D053614:Janus Kinase 2; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D011388:Prolactin / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011981:Receptors, Prolactin / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D013601:T-Lymphocytes; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D019000:Phosphotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2