PIN1與 Grb7交互作用之鑑定及分析

Abstract

Grb7(Growth Factor Receptor Bound Protein 7)為一參與在細胞中許多信息傳導途徑的adaptor protein，為Grb family的一員。其蛋白結構分為Proline-rich domain、GM domain以及SH2 domain三部分，通常藉由SH2 domain與其他蛋白間的交互作用來將上游的訊息傳遞下去，例如EGFR(epidermal growth factor receptor)或Eph receptors，Grb7會被上游kinase磷酸化，如focal adhesion kinase (FAK)活化，進而參與細胞中的integrin signaling pathway 及 cell migration。在許多癌細胞中，特別地，Grb7在乳癌細胞中會與HER2(Human Epidermal growth factor Receptor 2)有co-amplification的現象，並且會參與癌症細胞的migration及tumorigenesis。近來開始針對Grb7來當作癌症的治療標靶，然而對於Grb7本身的下游訊息傳導的了解並沒有很清楚。因此為了探討Grb7的訊息傳導，我們首先藉由Yeast two-hybrid篩選與Grb7會交互作用的下游蛋白，並對這些可能的蛋白以 Ingenuity Pathways Analysis系統來進行分析，預測這些蛋白彼此間可能的交互作用關係。而我們發現Grb7會與Pin1( peptidyl-prolyl cis/trans isomerase)在Yeast two-hybrid有大量結合的現。Pin1為一可以針對磷酸化後的蛋白進行cis/trans異構化的異構酶，可在細胞層次中調控蛋白的穩定、活化及構形。另外也有報導指出Pin1會在癌細胞中大量表現。在本研究中，藉由co- immunoprecipitation以及表現GST-Pin1蛋白後進行GST pull-down的實驗結果，我們確認了無論是在in vivo 或是in vitro的環境下，在乳癌細胞中Pin1的確是可以與Grb7進行結合。另外，對Grb7進行truncation mapping 以及site-directed mutagenesis，確認了Grb7上Pin1可能的進行結合的Ser/Thr-Pro motifs : S194A。更進一步，我們想了解Grb7與Pin1的相互作用是否會對於Grb7本身的蛋白穩定性、磷酸化或是下游的信息傳遞有所影響。同時，我們也想找出可能參與在此交互作用中的上游Ser/Thr kinase，以及此交互作用是否在乳癌細胞中，對Grb7所調控的細胞移動及增殖有影響。

The growth factor receptor-bound protein-7 (Grb7) is the prototype of the Grb7 adaptor protein family, originally identified as a binding partner of the cytoplasmic tail of the epidermal growth factor receptor. The molecular architecture of Grb7 includes an N-terminal Proline-rich domain, a middle GM region and a C-terminal SH2 domain. Grb7 transmits upstream signal in a tyrosine phosphorylation manner, in which its C-terminal SH2 domain enables interaction with phospho-tyrosine motif of varied signaling protein kinases, such as EGFR (epidermal growth factor receptor), Eph receptors, and non-receptor tyrosine kinases. Subsequently, Grb7 is phosphorylated by the upstream kinases, such as focal adhesion kinase (FAK), Src, EGFR, and leads to regulation of diverse cellular functions including cell migration. Consistently, Grb7 is frequently overexpressed in breast cancers in co-amplification with HER2 (human epidermal growth factor receptor 2), which usually correlates to poor clinical outcomes. In this current study, we further attempt to investigate signaling molecules acting downstream of Grb7. Herein, we found that PIN1 (peptidyl-prolyl cis/trans isomerase) is able to interact with Grb7 based on a yeast two-hybrid library screening. In addition, we analyzed the Grb7-mediated potential signaling pathways or interactome among the proteins obtained by the yeast two-hybrid screening. PIN1 is an isomerase that can modulate the conformation of its substrates in a post-phosphorylation manner, thereby influencing protein function, activity, and/or stability. Not surprisingly, numerous reports indicate that overexpression of PIN1 exhibits in different human cancers, including breast cancers. By co-immunoprecipitation and GST-Pin1 pull-down assay, we confirmed that the PIN1 bona fide interacts with Grb7 in breast cancer cells. Moreover, utilizing truncation mapping and site-directed mutagenesis approaches, we identified potential Ser/Thr-Pro motifs of Grb7, S194, responsibly bound to PIN1. Perspectively, we are currently investigating biochemical and functional effects of this interaction. For instance, we will examine the stability, phosphorylated status, localization as well as the upstream Ser/Thr kinase(s) of Grb7 involved in the interaction with PIN1. Lastly, the influence of cell migration and proliferation, especially its significance in tumorigenesis and/or progression, will also be elucidated attributed by the interaction between Grb7 and PIN1.