Coss D, Kuo C B, Yang L, Ingleton P, Luben R, Walker A M
Division of Biomedical Sciences, University of California, Riverside 92521, USA.
Endocrinology. 1999 Nov;140(11):5087-94. doi: 10.1210/endo.140.11.7104.
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.
我们之前已经证明，磷酸化的催乳素（PRL）可作为未修饰PRL对Nb2细胞增殖活性的拮抗剂。磷酸化PRL的一种分子模拟物，用天冬氨酸残基替代正常磷酸化的丝氨酸（丝氨酸179），具有相同的特性。由于阻断未修饰激素的增殖活性所需的磷酸化激素或天冬氨酸突变体的量不到其四分之一，因此我们研究了天冬氨酸突变体的高效能是否通过产生另一种干扰性的细胞内信号级联反应来实现。将Nb2细胞在37℃下分别暴露于5或500 ng/ml的人NIDDK PRL、野生型重组PRL（未修饰的PRL）或天冬氨酸突变体PRL（假磷酸化PRL）1、5或10分钟。在5 ng/ml和10分钟时，野生型重组PRL比NIDDK制剂对Janus激酶2（JAK 2）的激活作用更强。这与之前关于野生型激素具有更高增殖活性的报道一致，并且主要反映了NIDDK制剂中存在一些磷酸化激素。在500 ng/ml和10分钟时，饱和状态消除了两种制剂反应之间的任何差异。在两种浓度下，对天冬氨酸突变体均未观察到JAK 2激活。然而，对于天冬氨酸处理的细胞，信号转导和转录激活因子5（STAT 5）的激活与其他两组一样强烈。时间进程实验排除了天冬氨酸突变体诱导的STAT 5磷酸化是早期JAK 2激活结果的可能性。本研究中的实验还有趣地表明，在没有PRL且未检测到JAK 2或STAT 5磷酸化的情况下，JAK 2和STAT 5会预先结合。与JAK 2一样，天冬氨酸突变体不存在受体磷酸化。STAT 5a和STAT 5b激活的比较显示，天冬氨酸突变体诱导的STAT 5b磷酸化显著减少，同时STAT 5a - STAT 5b异二聚体也减少。然而，野生型和天冬氨酸突变体之间的STAT 5a激活没有区别。我们得出结论，无增殖活性的天冬氨酸突变体在不使用或极少使用JAK 2或受体磷酸化的情况下发出信号并激活STAT 5。另一方面，野生型具有增殖活性的PRL则利用受体磷酸化和JAK 2激活。
