title: Dissociation of Janus kinase 2 and signal transducer and activator of transcription 5 activation after treatment of Nb2 cells with a molecular mimic of phosphorylated prolactin.
abstract: We have previously demonstrated that phosphorylated PRL acts as an antagonist to the Nb2 proliferative activities of unmodified PRL. A molecular mimic of phosphorylated PRL, which substitutes an aspartate residue for the normally phosphorylated serine (serine 179), has the same properties. Because it takes less than one fourth the amount of phosphorylated hormone, or the aspartate mutant, to block the proliferative activity of unmodified hormone, we have investigated whether the high potency of the aspartate mutant is achieved by the production of an alternate and interfering intracellular signal cascade. Nb2 cells were exposed to 5 or 500 ng/ml human NIDDK PRL, wild-type recombinant PRL (unmodified PRL), or aspartate mutant PRL (pseudophosphorylated PRL) for 1, 5, or 10 min at 37 C. At 5 ng/ml and 10 min, wild-type recombinant PRL showed greater activation of Janus kinase 2 (JAK 2) than the NIDDK preparation. This is consistent with a previous report of higher proliferative activity for the wild-type hormone and is primarily a reflection of the presence of some phosphorylated hormone in the NIDDK preparation. At 500 ng/ml and 10 min, saturation eliminated any differences between responses to the two preparations. JAK 2 activation was not seen in response to the aspartate mutant at either concentration. Signal transducer and activator of transcription 5 (STAT 5) activation was, however, just as robust for the aspartate-treated cells as for the other two groups. Time course experiments eliminated the possibility that STAT 5 phosphorylation in response to the aspartate mutant was the result of JAK 2 activation at earlier time points. Experiments in the present study also interestingly showed preassociation of JAK 2 and STAT 5 in the absence of PRL and the absence of detectable phosphorylation of either JAK 2 or STAT 5. Like JAK 2, receptor phosphorylation was absent with the aspartate mutant. A comparison between STAT 5a and STAT 5b activation showed a marked reduction in STAT 5b phosphorylation in response to the aspartate mutant, with concomitant reduction in STAT 5a-STAT 5b heterodimers. STAT 5a activation, however, was indistinguishable between the wild-type and aspartate mutant. We conclude that the nonproliferative aspartate mutant signals and activates STAT 5 without, or with minimal, use of JAK 2 or receptor phosphorylation. The wild-type proliferative PRL, on the other hand, uses receptor phosphorylation and JAK 2 activation.
authors.affiliation: Division of Biomedical Sciences, University of California, Riverside 92521, USA.
authors.forename: D
authors.initials: D
authors.lastname: Coss
authors.forename: C B
authors.initials: CB
authors.lastname: Kuo
authors.forename: L
authors.initials: L
authors.lastname: Yang
authors.forename: P
authors.initials: P
authors.lastname: Ingleton
authors.forename: R
authors.initials: R
authors.lastname: Luben
authors.forename: A M
authors.initials: AM
authors.lastname: Walker
mesh_terms: D001224:Aspartic Acid; D002455:Cell Division / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D053614:Janus Kinase 2; D007700:Kinetics; D016399:Lymphoma, T-Cell; D008894:Milk Proteins; D018716:Molecular Mimicry; D016296:Mutagenesis; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011388:Prolactin / Q000737:chemistry / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000494:pharmacology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D001224:Aspartic Acid; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2