Role of JAK2-PAK1 interaction 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)。2008年，美国预计有40,480名妇女死于乳腺癌。80%的人类乳腺癌中检测到催乳素受体(PRLR)，并在乳腺癌细胞中过度表达。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对乳腺癌中的PRLR信号有更全面的了解。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酪氨酸的磷酸化也能增加细胞的运动能力。在这项授权中，我们建议检验依赖催乳素的PAK1的JAK2磷酸化调节PAK1活性的假说。激活的PAK1对靶蛋白的调节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体，调节肌动蛋白细胞骨架，增加细胞的运动性和侵袭性，介导细胞周期蛋白D1基因转录，影响人乳腺癌细胞的致瘤性。AIM1将确定JAK2磷酸化的PAK1在调节PRL依赖的肌动蛋白细胞骨架重排、细胞运动和侵袭性中的作用。AIM2将确定PAK1在调节PRL激活的细胞周期蛋白D1基因转录中的作用。最后，Aim3将确定PAK1的JAK2磷酸化是否影响体内人乳腺癌细胞的致瘤性。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。 公共卫生意义：催乳素(PRL)是1928年被发现的一种脑下垂体因子，能够刺激兔的乳腺发育和哺乳，以及鸽子的农作物奶生产。几年后，催乳素被命名为催乳素，这是基于它刺激牛奶产生的能力。已有300多种不同的生物活动归因于催乳素。这些生物学功能是由特定的膜受体介导的。这些受体是非酪氨酸激酶，它们通过受体招募并在配体结合时激活的相关蛋白来传递信号。其中一种是JAK2酪氨酸激酶。我们最近发现，另一种蛋白质-PAK1-是JAK2的一种新底物。我们发现JAK2与PAK1结合，并使PAK1更具活性。激活的PAK1有助于更好的细胞存活和细胞迁移。然而，这两种蛋白质是如何协同工作的，以及它们的确切作用机制尚不清楚。PRL也与乳腺癌有关。乳腺癌是女性最常见的癌症--近三分之一(30%)的女性癌症发生在乳房。根据美国女性目前的预期寿命，每9名女性中就有1人在有生之年患上乳腺癌--1960年，这一风险为每14人中就有1人患上乳腺癌。关于正常细胞成为癌细胞的分子生物学机制，仍有许多未知之处。我们的长期目标是了解PRL的作用和失调导致包括乳腺癌在内的人类疾病的分子机制。为此，我们开始分析JAK2和PAK1之间的关系。在目前的提案中，我们将研究JAK2和PAK1如何促进细胞迁移和细胞存活，以及哪些基因受这两种蛋白的调控。了解这些蛋白质是如何共同作用的，将有助于设计新的治疗方法，并可能设计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.