核糖核酸蛋白粒之K蛋白的結合磷酸激?和其他蛋白的純化與鑑定

Abstract

核糖核酸蛋白粒之K蛋白(hnRNP K)是一個具有多重功能的分子，它在基因轉錄過程中可以扮演轉錄抑制子(transcriptional repressor)的角色，以調控基因表現；又本身屬於RNP家族成員，也參與mRNA processing的過程是。並且因其可和一些信號傳遞分子結合，而被推測它可能也有傳遞信號或聯擊兩條不同信號傳遞路徑的功能。可以看出hnRNP K是一個功能複雜的分子。 hnRNP K能被某些kinase(s)進行磷酸化反應是早已被肯定的特性，它被認為是屬於serine/threonine kinase的受質，並藉由p95vav這個分子的結合，成為聯擊tyrosine kinase信號傳遞路徑的橋樑。在本篇文章中，筆者針對此特性，探討是何種kinase可以對其作用。並且確定hnRNP K本身亦可能屬於tyrosine kinase的受質。並且也是如同過去所被預測的serine/threonine kinase(Erk1)的受質。此外利用不同的kinase inhibitors處理，大致區分出它可能參與的信號傳遞路徑。同時在純化kinase的過程中，找出幾個能與hnRNP K相互作用的分子，並證實在離體的實驗裡hnRNP K確實能與這個分子(DDX1)交互作用。而hnRNP K被磷酸化後對其功能有何種改變則是進一步想要探討的方向。

hnRNP K is a versatile molecule which can interact with RNA, DNA, and multiple proteins, including the proto-oncogene Vav, Src-like tyrosine and inducible serine/threonine kinases, the transcription factor TBP, C/EBPβ and transcription repressor Zik1. The repressor activity of hnRNP K for C/EBPβ seems to be regulated through phosphorylation / dephosphorylation mechanisms. K protein kinase(s) was identified in whole cell extracts or nuclear extracts from K562 cells or mouse liver by GST-hnRNP K affinity chromatography and LC/MS/MS analysis. The results indicate that several kinases are involved in the phosphorylation of hnRNP K. Both tyrosine kinase(s) and Erk1 were shown to be able to phosphorylate hnRNP K. Several kinase inhibitors have been used to identify the specific K protein kinases. The involvement of these kinases in regulating hnRNP K by phosphorylation will be further studied. Multiple proteins have been detected from GST-hnRNP K affinity column chromatography. One of them, a putative RNA helicase, DDX1 has been identified. The functional implications of DDX1 and hnRNP K interaction are discussed.