探討syndecan-2和RACK1的交互作用與其細胞生理意義之研究

Abstract

在本實驗中，我們利用重組蛋白和親和管柱進行細胞外(in vitro)的實驗，發現重組syndecan-2和RACK1有交互作用，而在細胞內(in vivo)實驗，經免疫沉澱法和細胞免疫染色法也證實RACK1的確存在於syndecan-2蛋白複合體中，且兩分子的交互作用主要位於細胞膜上。進一步探討syndecan-2和RACK1的作用，我們發現當syndecan-2第180位置的酪胺酸在磷酸化狀態下，和RACK1的交互作用有增強的現象。 另一方面，我們以已知會造成syndecan-2酪氨酸磷酸化的細胞素(cytokine)，巨噬細胞群叢形成促進因子(GM-CSF)處理細胞，syndecan-2和RACK1的作用不論是有無磷酸化的參與(phosphate-dependent or independent)皆有增強的現象，且有時間和劑量上的效應(time-and dose-dependent effect)，該現象推測可能是與syndecan-2的磷酸化及syndecan-2的叢聚現象(clustering)所造成，但仍需更進一步實驗證實。 最後，我們試著分析syndecan-2在細胞生理的功能，並探討與RACK1交互作用的相關性，來進一步了解這兩個分子交互作用可能的細胞生理意義。由上述實驗結果，我們知道syndecan-2胞內區域磷酸化會增加兩分子間的作用，因此我們建構了正常型(wild type：Syn-2-wt)、酪胺酸單點突變(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F)及胞內區域(intracellular domain：PEP Syn-2-cyto) 的syndecan-2表現載體，在細胞中進行表現後，分別以細胞型態、貼附、爬移和增生等生理現象和生化方式進行觀察和分析，我們發現在不同載體表現的轉染細胞中，其細胞生理現象的差異和syndecan-2 /RACK1磷酸化參與交互作用的部分具有關聯性，然而是否藉由該作用的差異性來調控可能的分子(如：Src kinase or Protein kinase c)進而造成細胞生理現象的差異，其中的分子機轉仍須進一步的實驗證明。在本實驗中雖然無法明確了解syndecan-2和RACK1交互作用的意義，但仍提供了將來可能的實驗方向。

In this study, RACK1 (Receptor for Activated C Kinase 1) was found to be reactive with syndecan-2 in vitro and in vivo. Through affinity column chromatography and immunoprecipitation analysis as well as immunocytochemical colocalization studies, the reaction between RACK1 and syndecan-2 was evidenced in BALB/3T3 cells. Recombinant syndecan-2 and PEP Syn-2-cyto were applied to demonstrate that tyrosine 180 of syndecan-2 is a targeted site for Src tyrosine kinase and the reaction with RACK1 is enhanced after this tyrosine phosphorylation. In parallel, when granulocyte-macrophage colony-stimulating factor (GM-CSF) was applied to activate cellular tyrosine kinase of HeLa cells, a significant positive interaction was revealed between syndecan-2 and RACK1 with time and dose-dependence. HeLa cells were further subject to transfections with wild type and mutant syndecan-2 vectors(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F) to show that the reaction of syndecan-2 with RACK1 was suppressed when tyrosine 180 phosphorylation site was absent. To elucidate the physiological significance of this selective reaction between syndecan-2 and RACK1, studies with adhesion, migration, and poliferation were focused. HeLa cells transfected with Syn-2-Y180F mutant vectors exhibited less motile and adhesion ability. Furthermore, the morphology of Syn-2-Y180F transfants became round and refractile. These results imply that tyrosine 180 of syndecan-2 may involve in the cytoskeleton organization, focal contacts formation, and tyrosine kinase regulation through selective reaction with RACK1.