P13K/AKT crosstalk with ER-signaling and cell survival

描述(申请人提供)：这项研究的主要长期目标是了解AKT信号与雌激素受体(ER)的串扰在控制细胞存活和在抗雌激素耐药性发展中的作用。我们假设AKT通路的功能是靶向并磷酸化p160辅活化子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，AKT和雌激素信号在ER-CoA转录水平的融合在调节细胞存活和影响激素依赖的细胞系统(如乳腺癌)中的选择性雌激素受体调节剂(SERM)活性方面是至关重要的。我们提出的具体目标是：1)通过检测AKT对ER-AF2和/或p160 CoA GRIP的特异性靶向、磷酸化和激活来确定AKT-ER串扰的机制；2)通过检测AKT-ER-CoA对内源性雌激素反应基因的调节、SERM活性和基因表达的改变以及细胞的长期存活，来确定这种串扰的生理相关性。具体目标#1，确定AKT靶向和ER-AF2的磷酸化作为串扰机制的作用。我们建议确定ER-AF2靶向是否是通过AKT直接磷酸化发生的，以及特定的磷酸化位点在AKT-ER串扰中的作用。具体目的#2，暗示AKT调节p160辅活化子(GRIP)磷酸化是雌激素受体激活和募集的机制。研究将确定AKT是否通过特异性靶向GRIP-NRID(核受体相互作用域)或GRIP-AD2(激活域-2)来磷酸化和激活GRIP，作为AKT调节GRIP调控ER转录的机制。具体目的#3，确定在AKT介导的ER依赖的细胞存活、基因表达和SERM活性调节中对GRIP磷酸化/功能的需求。为此，我们将确定AKT诱导的ER-CoA募集/激活在调节细胞生存基因表达(Bcl2)中的作用以及AKT-ER-CoA信号传导对SERM活性的影响。这些研究还将确定AKT磷酸化ER或GRIP的机制是否在AKT-ER介导的细胞存活调节中起关键作用。作为一个整体，预计这一建议将建立由研究人员3K-AKT级联所使用的机制，以靶向ER和通过这一途径增强活性所需的ER转录复合体的后续成分。这种串扰和识别的机制将被用来建立该途径在细胞存活和改变的SERM活性方面的生物学相关性。

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.