Biochemical and Functional Characterization of Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein K Protein

• Main Authors: Chen, Huei-Mei, 陳慧美
• Other Authors: Chang, Ching-Jin
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/63417693315044910400

碩士 === 國立臺灣大學 === 生化科學研究所 === 91 === hnRNP K (Heterogeneous nuclear ribonucleoprotein K) was first discovered as a component of the hnRNP family. hnRNP K interacts with two different groups of protein partners, including proteins related to signal transduction (e.g. c-Src; Erk; Lck; PKC and Vav) and proteins related to gene regulation (e.g. C/EBP β; EF-1α; Zik-1). Besides interacting with proteins, hnRNP K also binds in a sequence-specific fashion to RNA and to single- or double-stranded DNA. Taken toghther, these interactions drive hnRNP K to regulate transcription, translation, pre-mRNA processing and mRNA transportation. Several particular domains has been identified in hnRNP K, including three KH domains (K homology) which links hnRNP K to proteins, RNA and DNA, a proline-rich domain in C terminal which binds hnRNP K to SH3 (Src homology 3) containing proteins, and NLS (nuclear localization signal) together with KNS (hnRNP K nuclear shuttling) responsible for hnRNP K shuttling in and out of nucleus. hnRNP K can be phosphorylated in vivo and in vitro. So far, it has been reported that hnRNP K is a substrate of both tyrosine kinases and ser/thr kinases. At least seven amino acids had been identified as possible phosphorylation sites. The interactions of hnRNP K with nucleic acids and protein partners along with the localization of hnRNP K itself are likely to be influenced by a chain of phosphorylation and dephosphorylation events. In this thesis, we focus on the influences of phosphorylation by ERK 2, a serine kinase. We applied the method of site directed mutagenesis to produce mutation constructs. Mutation at ERK phosphoacceptor sites mimics the unphosphorylated (ser to ala) and phosphorylated (ser to asp) forms of hnRNP K. We observed that phosphorylation drives cytoplasmic accumulation of hnRNP K by in vivo study. Also, we found that phosphorylation diminishes the binding activity of hnRNP K with pyrimidine-rich DNA, and regulates the expression of DICE (differentiational control element)-containing genes as well. These data suggest that phosphorylation is a crucial regulation event that controls the distribution of hnRNP K and many functions of hnRNP K in cell.