Role of JAK2-PAK1 interaction in prolactin-dependent signaling

描述（由申请方提供）：在正常乳腺发育中，激素催乳素（PRL）对肺泡增殖和分化至关重要。越来越多的证据支持PRL参与乳腺癌，乳腺癌是女性癌症的主要类型，也是女性癌症死亡的第二大原因（仅次于肺癌）。2008年，预计美国将有40，480名妇女死于乳腺癌。催乳素受体（PRLR）在80%的人类乳腺癌中被检测到，并在乳腺癌细胞中过度表达。正常和肿瘤乳腺上皮细胞合成催乳素和催乳素受体，因此催乳素可作为人乳腺癌细胞的自分泌生长因子。这些结果表明，需要更全面地了解乳腺癌中的PRLR信号。酪氨酸（Tyr）激酶JAK 2被鉴定为PRLR结合的信号分子。识别被招募到PRLR-JAK 2的蛋白质并剖析随后被激活的信号通路最终将为理解PRL作用提供基础。初步数据表明，丝氨酸-苏氨酸激酶PAK 1与JAK 2结合并被JAK 2磷酸化。二维肽图谱鉴定了PAK 1的三个Tyr（s），其被JAK 2磷酸化。JAK 2对PAK 1的Tyr磷酸化也显示出增加细胞运动性。在这项研究中，我们提出了一个假设，即催乳素依赖的JAK 2磷酸化PAK 1调节PAK 1的活性。活化的PAK 1调节靶蛋白可能依赖于磷酸化事件或/和蛋白质-蛋白质相互作用，导致形成多蛋白复合物，其调节肌动蛋白细胞骨架，增加细胞运动性和侵袭性，介导细胞周期蛋白D1基因转录并影响人乳腺癌细胞的致瘤性。Aim 1将决定JAK 2磷酸化PAK 1在调节PRL依赖性肌动蛋白细胞骨架重排、细胞运动性和侵袭性中的作用。Aim 2将决定PAK 1在调节PRL激活的细胞周期蛋白D1基因转录中的作用。最后，Aim 3将确定PAK 1的JAK 2磷酸化是否影响体内人乳腺癌细胞的致瘤性。由于PAK 1和PRL都与乳腺癌有关，因此拟议的研究可能最终填补上游PRL-PRLR-JAK 2事件和下游PAK 1依赖性功能之间的现有空白，以了解人类乳腺癌的机制。JAK 2对PAK 1的Tyr磷酸化可能代表了寻找人类乳腺癌病因和治疗的新分子靶点。 公共卫生相关性：催乳素（PRL）于1928年被发现是一种脑垂体因子，能够刺激兔子的乳腺发育和泌乳，以及鸽子的农作物奶的生产。几年后，催乳素这个名字被赋予，基于它刺激牛奶生产的能力。PRL具有300多种不同的生物活性。这些生物学功能由特定的膜受体介导。这些受体是非酪氨酸激酶，它们通过由受体募集并在配体结合时激活的相关激酶来传递信号。这些激酶之一是JAK 2酪氨酸激酶。我们最近发现，另一种蛋白质-PAK 1-是JAK 2的一种新底物。我们发现JAK 2与PAK 1结合，使PAK 1更活跃。活化的PAK 1有助于更好的细胞存活和细胞迁移。然而，这两种蛋白质如何协同工作以及它们的确切作用机制尚不清楚。PRL也与乳腺癌有关。乳腺癌是女性中最常见的癌症-近三分之一（30%）的女性癌症发生在乳房中。根据目前美国女性的预期寿命，每9名女性中就有1名会在一生中患上乳腺癌，而在1960年，这一风险是每14名女性中就有1名。关于正常细胞转变为癌细胞的分子生物学机制仍有很多未知之处。我们的长期目标是了解PRL作用的分子机制，以及导致人类疾病（包括乳腺癌）的PRL失调。为了实现这一目标，我们开始分析JAK 2和PAK 1之间的关系。在目前的提案中，我们将研究JAK 2和PAK 1如何增加细胞迁移和细胞存活，以及哪些基因受这两种蛋白质的调控。了解这些蛋白质如何协同工作将有助于设计新的治疗方法，并可能设计用于治疗不同人类疾病和乳腺癌的药物。

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 2008, 40,480 women are expected to die from breast cancer in the U.S. 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 in breast cancer. 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 increase cell motility. In this grant we propose to examine the hypothesis prolactin-dependent JAK2 phosphorylation of PAK1 regulates PAK1 activity. Activated PAK1 regulation of target proteins may depend on phosphorylation events or/and protein-protein interactions, leading to the formation of a multiprotein complex that modulates the actin cytoskeleton, increases cell motility and invasiveness, mediates cyclin D1 gene transcription and affects tumorigenicity of human breast cancer cells. Aim1 will determine the role of JAK2-phosphorylated PAK1 in regulating PRL-dependent actin cytoskeleton rearrangement, cell motility and invasiveness. Aim2 will determine the role of PAK1 in regulating PRL-activated cyclin D1 gene transcription. Finally, Aim3 will determine whether JAK2 phosphorylation of PAK1 affects the tumorigenicity of human breast cancer cells in vivo. 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. PUBLIC HEALTH RELEVANCE: Prolactin (PRL) was discovered in 1928 as a pituitary factor able to stimulate mammary gland development and lactation in rabbits, as well as the production of crop milk in pigeons. A few years later, the name prolactin was given, based on its ability to stimulate milk production. More than 300 separate biological activities have been attributed to PRL. These biological functions are mediated by specific membrane receptors. These receptors are non tyrosine kinases, they transduce the signal via associated kinases that are recruited by the receptor and activated upon ligand binding. One of these kinases is JAK2 tyrosine kinase. We have recently shown that another protein - PAK1 - is a novel substrate of JAK2. We showed that JAK2 binds to PAK1 and makes PAK1 more active. Activated PAK1 contributes to better cell survival and cell migration. However, how these two proteins work together and the precise mechanism of their action is unknown. PRL is also involved in breast cancer. Breast cancer is the most commonly diagnosed cancer in women - nearly 1 in 3 (30%) of all cancers in women occur in the breast. Based on the current life expectancy for women in the United States, 1 out of 9 women will develop breast cancer in her lifetime - a risk that was 1 out of 14 in 1960. Much is still unknown regarding the molecular biological mechanism by which a normal cell becomes a cancer cell. Our long term goal is to understand the molecular mechanisms of PRL action and disregulation of which leads to human diseases including breast cancer. Toward this aim, we have started to analyze the relationship between JAK2 and PAK1. In the current proposal we will study how JAK2 and PAK1 increase cell migration and cell survival, and which genes are regulated by these two proteins. Understanding how these proteins work together will help to design new therapeutic approaches and possibly drugs for treatment of different human diseases and breast cancer.