TARGETED PROTEOMIC ANALYSIS OF 14-3-3 SIGMA, A P53 EFFECTOR COMMONLY SILENCED I

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。为了全面鉴定体内与14-3-3 sigma相互作用的蛋白，我们将串联亲和纯化和多维蛋白鉴定技术相结合，对人体细胞中117个与14-3-3 sigma相关的蛋白进行了表征。大多数已鉴定的蛋白含有一个或几个可磷酸化的14-3-3结合位点，表明可能与14-3-3 sigma直接相互作用。25个蛋白之前没有被指定任何功能，并被命名为SIP2-26 （14-3-3 sigma相互作用蛋白）。在已知功能的92种相互作用物中，有许多蛋白先前与致癌信号传导（APC, a - raf， B-RAF和c-RAF）和细胞周期调节（AJUBA, c-TAK， ptv -1和WEE1）有关。最大的功能类别包括参与调节细胞骨架动力学、极性、粘附、有丝分裂信号和运动的蛋白质。因此，在损伤实验中，14-3-3 sigma异位表达阻止了细胞迁移，并增强了丝裂原激活的蛋白激酶信号传导。所鉴定蛋白的功能多样性表明，14-3-3 sigma的诱导除了先前描述的对G2/M进展的抑制作用外，还可以使p53影响许多过程。这些数据表明，由于表观遗传沉默或p53突变导致14-3-3 sigma表达的癌症特异性缺失，可通过多种途径促进癌症的形成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.