Role of JAK2-PAK1 interaction in prolactin-dependent signaling

描述（申请人提供）：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与乳腺癌有关，乳腺癌是妇女的主要癌症类型，也是妇女癌症死亡的第二大原因（仅次于肺癌）。2008年，美国预计将有40,480名女性死于乳腺癌。泌乳素受体（PRLR）在80%的人类乳腺癌中被检测到，并且在乳腺癌细胞中过度表达。正常和肿瘤乳腺上皮细胞可合成PRL和PRLR， PRL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JAK2被鉴定为prlr结合的信号分子。鉴定PRLR-JAK2募集的蛋白和随后被激活的信号通路的解剖将最终为理解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磷酸化Tyr相关。二维肽图谱鉴定出PAK1中被JAK2磷酸化的三个Tyr(s)。JAK2对PAK1的Tyr磷酸化也显示出增加细胞运动性。在本研究中，我们提出检验催乳素依赖性JAK2磷酸化PAK1调控PAK1活性的假设。活化的PAK1对靶蛋白的调控可能依赖于磷酸化事件或/和蛋白-蛋白相互作用，导致形成多蛋白复合物，调节肌动蛋白细胞骨架，增加细胞活力和侵袭性，介导cyclin D1基因转录并影响人乳腺癌细胞的致瘤性。ai1将决定jak2磷酸化PAK1在调节prl依赖性肌动蛋白细胞骨架重排、细胞运动和侵袭性中的作用。Aim2将决定PAK1在调节prl激活的cyclin D1基因转录中的作用。最后，Aim3将在体内确定JAK2磷酸化PAK1是否会影响人乳腺癌细胞的致瘤性。由于PAK1和PRL都与乳腺癌有关，这些研究可能最终填补我们对人类乳腺癌机制理解中上游PRL- prlr - jak2事件与下游PAK1依赖功能之间的空白。JAK2对PAK1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。

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.