Role of the serine-threonine kinase PAK1 in prolactin-dependent signaling

描述(申请人提供)：在正常的乳房发育中，催乳素(PRL)对肺泡的增殖和分化至关重要。越来越多的证据支持PRL与乳腺癌有关，乳腺癌是女性的主要癌症类型，也是女性癌症死亡的第二大原因(仅次于肺癌)。2002年，预计将诊断出203500例新的乳腺癌病例，预计将有39600名妇女死于这种疾病。催乳素受体(PRLR)在80%的人类乳腺癌中检测到，并且在乳腺癌细胞中过表达。正常乳腺上皮细胞和肿瘤乳腺上皮细胞合成PRL和PRLR，因此PRL可作为人乳腺癌细胞的自分泌生长因子。这些结果表明，有必要更全面地了解PRLR信号转导促进生长、抗细胞凋亡的途径。酪氨酸激酶JAK2是一种与PRLR结合的信号分子。鉴定招募到PRLR-JAK2中的蛋白质并剖析随后被激活的信号通路最终将为理解PRL的作用提供基础。初步数据表明，丝氨酸苏氨酸激酶PAK1与JAK2结合，并被JAK2磷酸化。二维肽图谱鉴定了三个被JAK2磷酸化的PAK1Tyr(S)。JAK2对PAK1的酪氨酸磷酸化也显示出保护细胞免于凋亡的作用。这项资助建议检验这样的假设，即PAK1是JAK2的底物，并且作为对PRL的反应，PAK1被JAK2依赖的Tyr磷酸化激活并提高PRL依赖的细胞存活。AIM1将在体内验证PRL促进PAK1的Tyr磷酸化。AIM2将确定PAK1的JAK2磷酸化是否改变了PAK1的激酶活性和/或PAK1结合PAK1靶标的能力。在Aim3中，将确定PAK1的JAK2 Tyr磷酸化对PRL依赖的细胞存活的影响。由于PAK1和PRL都参与了乳腺癌的发生，因此本研究可能最终填补上游的PRL-PRLR-JAK2事件和下游的PAK1依赖的功能在我们理解人类乳腺癌发生机制方面的空白。JAK2对PAK1的酪氨酸磷酸化可能是探索人类乳腺癌病因和治疗的一个新的分子靶点。

DESCRIPTION (provided by applicant): In normal mammary development, the hormone prolactin (PRL) is critical for alveolar proliferation and differentiation. Increasing evidence supports the involvement of PRL in breast cancer, the leading type of cancer in women and the second leading cause (after lung cancer) of cancer death among women. In 2002, 203,500 new cases of breast cancer were expected to be diagnosed, and 39,600 women were expected to die of the disease. The prolactin receptor (PRLR) is detected in 80% of human breast cancers, and is overexpressed in breast cancer cells. Normal and tumor mammary epithelial cells synthesize PRL and PRLR, thus the PRL could behave as an autocrine growth factor for human breast cancer cells. These results suggest the need for a more complete understanding of PRLR signaling to growth promoting, anti-apoptotic pathways. Tyrosine (Tyr) kinase JAK2 was identified as a PRLR-bound signaling molecule. Identification of the proteins recruited to the PRLR-JAK2 and dissection of the signaling pathways that are subsequently activated will ultimately provide a basis for understanding PRL action. Preliminary data demonstrate that the serine-threonine kinase PAK1 associates with and is Tyr phosphorylated by JAK2. Two-dimensional peptide mapping identified three Tyr(s) of PAK1 which are phosphorylated by JAK2. Tyr phosphorylation of PAK1 by JAK2 was also shown to protect cells from apoptosis. This grant proposes to examine the hypothesis that PAK1 is a substrate for JAK2 and that in response to PRL, PAK1 is activated by JAK2-dependent Tyr phosphorylation and enhances PRL-dependent cell survival. Aim1 will verify that PRL promotes Tyr phosphorylation of PAK1 in vivo. Aim2 will determine whether JAK2 phosphorylation of PAK1 alters PAK1 kinase activity and/or ability of PAK1 to bind PAK1 targets. In Aim3 the effect of JAK2 Tyr phosphorylation of PAK1 on PRL-dependent cell survival will be determined. Because both PAK1 and PRL have been implicated in breast cancer, the proposed studies may ultimately fill out the existing gap between upstream PRL-PRLR-JAK2 events and downstream PAK1-dependent functions in our understanding of the mechanism of human breast cancer. Tyr phosphorylation of PAK1 by JAK2 is likely to represent a novel molecular target in the search for the etiology and treatment of human breast cancer.