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p160共激活因子GRIP和ER蛋白并使其磷酸化，从而调控辅酶a的募集、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转录水平上的趋同对细胞存活的调节至关重要，并影响乳腺癌等激素依赖细胞系统中选择性雌激素受体调节剂（SERM）的活性。本文提出的具体目标是：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对内源性雌激素应答基因的调节、serm活性和基因表达的改变以及细胞长期存活的影响来确定这种串扰的生理相关性。特异性目的1：确定AKT靶向和ER-AF2磷酸化作为串扰机制的作用。我们建议通过AKT的直接磷酸化以及AKT- er串扰中特定磷酸化位点的作用来确定ER-AF2靶向是否发生。目的2：揭示AKT调控p160共激活因子-(GRIP)磷酸化作为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（核受体相互作用结构域）或GRIP- ad2（激活结构域-2）磷酸化并激活GRIP，作为AKT调控GRIP控制ER转录的机制。具体目的#3，确定GRIP磷酸化/功能在akt介导的er依赖性细胞存活、基因表达和SERM活性调节中的需求。为此，我们将确定特异性AKT诱导的ER-CoA募集/激活在调节细胞存活基因（Bcl-2）表达中的作用，以及AKT-ER-CoA串导对SERM活性的影响。这些研究还将确定akt磷酸化ER或GRIP的机制在AKT-ER介导的细胞存活调节中是否至关重要。总体而言，我们预计该提案将建立研究者3k - akt级联所使用的机制，以靶向内质网和内质网转录复合体的后续组分，这些组分需要通过该途径增强活性。这种串扰和确定的机制将用于确定该途径在细胞存活和改变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.