Regulation of Breast Cancer Growth by MLK-3

描述(由申请人提供)： 摘要/综述原理：蛋白激酶在细胞功能中的作用，包括生长、分化和细胞死亡，是公认的。同源蛋白激酶对蛋白质的磷酸化是一个动态过程，它深刻影响细胞中蛋白质的生理功能。在包括癌症在内的疾病状态下，这一过程可能会变得不受调控，人们试图将某些蛋白激酶作为靶点用于乳腺癌(和其他癌症)的治疗。本实验室的重点是确定一个新的蛋白激酶家族成员的生理功能，称为混合血统蛋白激酶(MLKs)。MLK家族成员的独特之处在于，它们可以针对丝氨酸/苏氨酸和酪氨酸残基进行磷酸化。这个新的激酶家族在肿瘤发生中的作用还不是很清楚。我们已经做了最初的观察，MLK3直接与一种名为Pin1的蛋白质相互作用并磷酸化。已知Pin1在乳腺肿瘤中高表达，并促进乳腺癌细胞的存活和增殖。Pin1是肽基脯氨酰顺/反式异构酶家族的成员，它与底物结合，异构化特定的pSer/Thr-Pro基序，催化构象变化。这种构象变化可以对Pin1底物的作用产生深远的影响，从而显著影响细胞功能。然而，Pin1促进乳腺癌发生的机制尚不清楚。我们观察到，Pin1直接被MLK3在一个非常特定的残基上磷酸化，导致其异构酶活性激活(Pin1功能增强的霍尔标志)，这可能在其致瘤功能中起重要作用。我们的观察结果支持这一点，即磷酸化Pin1仅定位于高侵袭性乳腺癌细胞系的细胞核中，并促进细胞周期的G2/M转变。这些观察结果表明，磷酸化的Pin1可能在细胞核中具有促进细胞周期进展的功能靶点(S)。由于Pin1的几个靶点存在于细胞核中，并可能参与肿瘤的发生过程，我们的结果将通过专门识别细胞核中的磷酸化Pin1的靶点及其在乳腺癌中的潜在作用来帮助我们了解这一知识。假设：根据我们的新发现，Pin1被MLK3直接磷酸化并激活，我们假设磷酸化的Pin1靶向细胞核中的特定底物(S)，这可能有助于促进乳腺癌的发生。目的：针对上述假设，我们提出了三个具体目标：(1)研究Pin1的磷酸化，确定其靶标(S)/底物(S)。(2)确定Pin1蛋白(通过MLK3)磷酸化在乳腺癌发病机制中的功能后果。(3)明确MLK3基因的药理学干扰或基因干扰是否抑制了Pin1在乳腺癌发病机制中的病理作用。使用程序：这些研究将采用广泛的技术方法，包括特定RNAi/shRNA的基因沉默、免疫沉淀、激酶分析、Western blotting、细胞增殖和细胞死亡分析。研究模型将包括培养的细胞和小鼠乳腺癌的异种移植。潜在新发现的意义：这些研究的结果将首次阐明MLK3调节Pin1磷酸化及其在乳腺癌发病过程中功能的详细机制。此外，我们的结果将指出利用MLK抑制剂(S)作为一种新的乳腺癌治疗干预措施的可能性。

DESCRIPTION (provided by applicant): Abstract/Summary Rationale: Role of protein kinases in cell functions including growth, differentiation, and cell death is well established. Phosphorylation of proteins by cognate protein kinases is a dynamic process known to profoundly influence the physiological function of proteins in the cell. This process can become dysregulated in disease states including in cancer, and attempts have been made to target certain protein kinases for therapy of breast cancer (and other cancers). The focus of this laboratory is to define the physiological functions of a novel member of a protein kinase family, called Mixed Lineage Kinases (MLKs). MLK family members are unique in the sense that they can target both Ser/Thr and tyrosine residues for phosphorylation. The function of this novel family of kinases in tumorigenesis is not well known. We have made the original observation that MLK3 directly interacts with and phosphorylates a protein called Pin1. Pin1 is known to be highly expressed in breast tumors and promotes survival and proliferation of breast cancer cells. Pin1 is a member of peptidyl prolyl cis/trans isomerase family, which binds to its substrates and isomerizes specific pSer/Thr-Pro motifs and catalytically induces conformational changes. Such conformational changes can have profound effects on the role of Pin1 substrates so as to significantly impact cellular functions. However, the mechanism by which Pin1 promotes breast cancer pathogenesis is unclear. Our observation that Pin1 is directly phosphorylated by MLK3 at a very specific residue resulting in the activation of its isomerase activities (a hall mark of increased Pin1 function) may be important in its tumorigenic function. This is supported by our observation that phospho-Pin1 is exclusively localized in the nucleus of a highly aggressive breast cancer cell line and promotes G2/M transition of cell cycle. These observations suggest that phosphorylated Pin1 might have functional target(s) in the nucleus to promote cell cycle progression. Since several targets of Pin1 exist in the nucleus and may contribute to the oncogenic process, our results would contribute to this knowledge by specifically identifying targets of the phospho-Pin1 in the nucleus and their potential role in breast cancer. Hypothesis: Based on our novel finding that Pin1 is directly phosphorylated and activated by MLK3, we hypothesize that phosphorylated Pin1 targets specific substrate(s) in the nucleus which may serve to promote breast cancer pathogenesis. Objectives: To address the above hypothesis, we propose three Specific Aims: (1) To characterize the phosphorylation of Pin1 and identify the target(s)/substrate(s) of phospho-Pin1. (2) To determine the functional consequences of Pin1 phosphorylation (by MLK3) in breast cancer pathogenesis. (3) To determine whether pharmacological or genetic disruption of MLK3 inhibits the pathological functions of Pin1 in breast cancer pathogenesis. Procedures to be used: These studies will employ a broad range of technical approaches, including gene silencing by specific RNAi/ShRNA, immunoprecipitation, kinase assays, Western blotting, cell proliferation and cell death assays. Study models will include cultured cells and xenografts of breast cancer in mice. Significance of potential new findings: The results of these studies will elucidate, for the first time, detailed mechanism by which MLK3 regulates Pin1 phosphorylation and modulates its function during breast cancer pathogenesis. Further, our results will point to the possibility of utilizing MLK inhibitor(s) as a novel therapeutic intervention for breast cancer therapy.