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

We have previously demonstrated that phosphorylated PRL acts as an antagoni st to the Nb2 proliferative activities of unmodified PRL. A molecular mimic of phosphorylated PRL, which substitutes an aspartate residue for the norm ally phosphorylated serine (serine 179), has the same properties. Because i t takes less than one fourth the amount of phosphorylated hormone, or the a spartate mutant, to block the proliferative activity of unmodified hormone, we have investigated whether the high potency of the aspartate mutant is a chieved by the production of an alternate and interfering intracellular sig nal cascade. Nb2 cells were exposed to 5 or 500 ng/ml human NIDDK PRL, wild -type recombinant PRL (unmodified PRL), or aspartate mutant PRL (pseudophos phorylated PRL) for 1, 5, or 10 min at 37 C. At 5 ng/ml and 10 min, wild-ty pe 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 reflec tion of the presence of some phosphorylated hormone in the NIDDK preparatio n. At 500 ng/ml and 10 min, saturation eliminated any differences between r esponses to the two preparations. JAK 2 activation was not seen in response to the aspartate mutant at either concentration. Signal transducer and act ivator of transcription 5 (STAT 5) activation was, however,just as robust f or the aspartate-treated cells as for the other two groups. Time course exp eriments 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 preasso ciation of JAK 2 and STAT 5 in the absence of PRL and the absence of detect able phosphorylation of either JAK 2 or STAT 5. Like JAK 2, receptor phosph orylation was absent with the aspartate mutant. A comparison between STAT 5 a and STAT 5b activation showed a marked reduction in STAT 5b phosphorylati on 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 nonprolif erative aspartate mutant signals and activates STAT 5 without, or with mini mal, use of JAK 2 or receptor phosphorylation. The wild-type proliferative PRL, on the other hand, uses receptor phosphorylation and JAK 2 activation.