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之前的资助期内发现的新型磷酸酶PHLPP （PH结构域富亮氨酸重复蛋白磷酸酶；发音为“flip”）介导的信号终止的分子和细胞机制。该建议的核心假设是，PHLPP终止由PDK- 1开启的信号通路，特别是Akt信号通路，并且终止的特异性是通过亚细胞定位和大分子相互作用实现的。1. PHLPP的分子机制：本节的目的是了解PP2C家族新成员PHLPP的酶学和生物化学。具体来说，我们将研究三种PHLPP同工酶的动力学和底物特异性，交替剪接的PHLPP1 (a和（）和PHLPP2，并在Aims 2和3中开发细胞研究工具。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。我们将验证PHLPP同工异构体的PDZ结合基元选择性地将它们与特定的NHERF PDZ结构域蛋白连接在一起，形成一个支架网络，从而有效控制Akt信号的振幅和持续时间。我们将描述细胞中额外的PHLPP结合伙伴，并使用新的成像技术，我们将实时测量活细胞中的PHLPP活性。3. PHLPP在疾病中的作用：本研究旨在探讨PHLPP在人类癌症中的作用。PHLPP1和PHLPP2基因分别位于结肠癌和乳腺癌中最常缺失的染色体位点上。PHLPP同工酶在控制Akt信号振幅方面的功能使其成为这些位点上难以捉摸的肿瘤抑制因子的主要候选者。为了验证这一点，我们将筛选人类肿瘤中PHLPP的突变，研究PHLPP在乳腺癌细胞迁移中的作用，并建立小鼠模型来研究PHLPP基因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.