P13K/AKT crosstalk with ER-signaling and cell survival

描述（由申请人提供）：本研究的主要长期目标是了解AKT信号传导与雌激素受体（ER）的串扰在控制细胞存活和抗雌激素抗性发展中的作用。我们假设AKT通路的功能是靶向和磷酸化p160辅激活因子GRIP和ER蛋白，从而调节CoA募集、受体激活和基因表达。我们进一步假设，收敛的AKT和雌激素信号在ER-CoA转录水平是至关重要的细胞存活的调节和影响选择性雌激素受体调节剂（SERM）在激素依赖性细胞系统，如乳腺癌的活动。具体目的是：1）通过检查AKT对ER-AF 2和/或p160 CoA GRIP的特异性靶向、磷酸化和活化，确定AKT-ER串扰的机制; 2）通过检查AKT-ER-CoA对内源性雌激素应答基因的调节、SERM-activity和基因表达的改变以及长期细胞存活的影响，确定该串扰的生理相关性。具体目标#1，确定ER-AF 2的AKT靶向和磷酸化作为串扰机制的作用。我们建议确定ER-AF 2靶向是否通过AKT直接磷酸化发生以及特定磷酸化位点在AKT-ER串扰中的作用。具体目标#2，提示AKT调节p160辅激活因子（GRIP）磷酸化是激活和募集雌激素受体的机制。研究将确定AKT是否通过特异性靶向GRIP-NRID（核受体相互作用结构域）或GRIP-AD 2（激活结构域-2）磷酸化并激活GRIP，作为AKT调节GRIP控制ER转录的机制。具体目标#3，确定在ER依赖性细胞存活、基因表达和SERM活性的AKT介导的调节中对GRIP磷酸化/功能的需求。为此，我们将确定特定AKT诱导的ER-CoA募集/激活在细胞生存基因表达（Bcl-2）调节中的作用以及AKT-ER-CoA串扰对SERM活性的影响。这些研究还将确定上述ER或GRIP的AKT磷酸化机制是否在AKT-ER介导的细胞存活调节中至关重要。总体而言，预期该提议将建立由pH 3 K-AKT级联使用的机制，以靶向ER和增强该途径活性所需的ER转录复合物的后续组分。这种串扰和确定的机制将用于建立细胞存活和改变的SERM活性方面的途径的生物学相关性。

DESCRIPTION (provided by applicant): The primary long-term objective of this research is to understand the role of AKT-signaling cross-talk with the estrogen receptor (ER) in the control of cell survival and in the development of anti-estrogen resistance. We hypothesize that the AKT pathway functions to target and phosphorylate the p160 coactivator GRIP and ER proteins leading to regulation of CoA-recruitment, receptor-activation and gene expression. We further hypothesize that convergence of the AKT and estrogen signaling at the ER-CoA-transcriptional level is critical in the regulation of cell survival and influences selective estrogen receptor modulator (SERM) activity in hormone dependent cell systems such as breast carcinoma. The Specific Aims are proposed to 1) Determine the mechanisms of AKT-ER cross-talk by examining specific targeting, phosphorylation and activation of ER-AF2 and/or the p160 CoA GRIP by AKT and 2) Determine the physiological relevance of this cross-talk by examining effects of AKT-ER-CoA on the regulation of endogenous estrogen-responsive genes, alteration of SERM-activity and gene expression, and long-term cell survival. Specific Aim #1, To determine the role of AKT targeting and phosphorylation of ER-AF2 as a mechanism for cross-talk. We propose to determine if the ER-AF2 targeting occurs through direct phosphorylation by AKT and the role of specific phosphorylation sites in AKT-ER cross-talk. Specific Aim #2, To implicate AKT regulation of p160 coactivator-(GRIP) phosphorylation as a mechanism for activation of and recruitment to estrogen receptors. Studies will determine if AKT phosphorylates and activates GRIP though specific targeting of the GRIP-NRID (nuclear receptor interaction domain) or GRIP-AD2 (activation domain-2) as mechanisms for AKT regulation of GRIP in control of ER transcription. Specific Aim #3, to determine the requirement for GRIP phosphorylation/function in AKT-mediated regulation of ER-dependent cell survival, gene expression and SERM activity. In this aim, we will determine the role for specific AKT induced ER-CoA recruitment/activation in the regulation of cell survival gene expression (Bcl-2) and the influence of AKT-ER-CoA cross-talk on SERM activity. These studies will also determine if the mechanisms for AKT-phosphorylation of ER or GRIP from above are critical in the AKT-ER mediated regulation of cell survival. It is expected as a whole that this proposal will establish the mechanisms used by the investigator3K-AKT cascade to target the ER and the subsequent components of the ER transcription complex required for potentiation of activity by this pathway. This cross-talk and the mechanisms identified will be used to establish the biological relevancy of the pathway in terms of cell survival and altered SERM activity.