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

描述(由申请人提供)：这项提案的长期目标是了解我们在前一个资助期发现的新型磷酸酶PHLPP(PH域富含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。支持这一提议的中心假设是，PHLPP终止由PDK-1启动的信号通路，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：本部分的目的是了解PHLPP这一新的PP2C家族成员的酶学和生物化学。具体地说，我们将研究三种PHLPP同工酶的动力学和底物特异性，即可选择性剪接的PHLPP1(a和())和PHLPP2，并在AIMS 2和3.2中开发细胞研究工具。2.PHLPP的细胞机制：本节的目标是了解控制PHLPP在细胞中功能的机制。我们将测试这一假设，即PHLPP异构体的PDZ结合基序选择性地将它们与特定的NHERF PDZ结构域蛋白捆绑在一起，形成一个支架网络，允许有效控制Akt信号的幅度和持续时间。我们将鉴定细胞中更多的PHLPP结合伙伴，并使用新的成像技术，我们将在活细胞中实时测量PHLPP的活性。3.PHLPP在疾病中的作用：这一目标涉及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见的缺失染色体上。PHLPP同工酶在控制Akt信号幅度方面的功能使它们成为这些基因座上隐藏的难以捉摸的肿瘤抑制基因的主要候选者。为了测试这一点，我们将筛查人类肿瘤中PHLPP的突变，解决PHLPP在乳腺癌细胞细胞迁移中的作用，并开发一个小鼠模型，以解决PHLPP的基因缺失如何导致细胞生存和增殖的异常。

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.