Signal Termination by PHLPP, PH domain Leucine-rich repeat Protein Phosphatase

描述（由申请人提供）：本提案的长期目标是了解我们在前一资助期发现的新型磷酸酶PHLPP（PH结构域富含亮氨酸重复蛋白磷酸酶;发音为“翻转”）介导的信号终止的分子和细胞机制。推动这一提议的中心假设是PHLPP终止由PDK- 1开启的信号传导途径，特别是Akt信号传导途径，并且终止的特异性通过亚细胞定位和大分子相互作用实现。1. PHLPP的分子机制：本节的目的是了解PHLPP的酶学和生物化学，一个新的PP 2C家族成员。具体而言，我们将研究三种PHLPP同工酶的动力学和底物特异性，即选择性剪接的PHLPP 1（a和（））和PHLPP 2，并开发用于目标2和3的细胞研究的工具。2. PHLPP的细胞机制：本节的目的是了解控制细胞中PHLPP功能的机制。我们将测试PHLPP亚型的PDZ结合基序选择性地将它们与特定的NHERF PDZ结构域蛋白质连接，形成一个支架网络，允许有效控制Akt信号传导的幅度和持续时间的假设。我们将表征细胞中额外的PHLPP结合伴侣，并使用新的成像技术，我们将在活细胞中真实的时间测量PHLPP活性。3. PHLPP在疾病中的作用：这一目标解决了PHLPP在人类癌症中的作用。PHLPP 1和PHLPP 2基因分别位于据报道在结肠癌和乳腺癌中最常缺失的染色体基因座上。PHLPP同工酶在控制Akt信号传导幅度中的功能使其成为这些位点上隐藏的难以捉摸的肿瘤抑制因子的主要候选者。为了测试这一点，我们将筛选人类肿瘤中PHLPP的突变，解决PHLPP在乳腺癌细胞中细胞迁移中的作用，并开发小鼠模型来解决PHLPP的遗传缺失如何导致细胞存活和增殖异常。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the molecular and cellular mechanisms of signal termination mediated by the novel phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase; pronounced 'flip') that we discovered in the preceding funding period. The central hypothesis driving this proposal is that PHLPP terminates signaling pathways that are turned on by PDK- 1, notably Akt signaling pathways, and that specificity in termination is achieved by subcellular location and macromolecular interactions. 1. Molecular Mechanisms of PHLPP: The goal of this section is to understand the enzymology and biochemistry of PHLPP, a new PP2C family member. Specifically, we will examine the kinetics and substrate specificity of the three PHLPP isozymes, the alternatively spliced PHLPP1 (a and () and PHLPP2, and develop tools for cellular studies in Aims 2 and 3. 2. Cellular Mechanisms of PHLPP: The goal of this section is to understand the mechanisms that control the function of PHLPP in cells. We will test the hypothesis that the PDZ binding motifs of the PHLPP isoforms selectively tether them to specific NHERF PDZ domain proteins, forming a scaffolding network that allows effective control of the amplitude and duration of Akt signaling. We will characterize additional PHLPP binding partners in cells and, using novel imaging technologies, we will measure PHLPP activity in real time in live cells. 3. PHLPP in disease: This aim addresses the role of PHLPP in human cancers. The PHLPP1 and PHLPP2 genes are located on the chromosomal loci reported to be the most commonly deleted in colon cancer and breast cancer, respectively. The function of PHLPP isozymes in controlling the amplitude of Akt signaling poises them as prime candidates to be the elusive tumor suppressors harbored on these loci. To test this, we will screen human tumors for mutations in PHLPP, address the role of PHLPP in cell migration in breast cancer cells, and develop a mouse model to address how genetic deletion of PHLPP could lead to abnormalities in cell survival and proliferation.