Kinetic analysis and ubiquitination sites prediction of Src kinase

• Main Authors: Hung, Hsiu-Yi, 洪秀怡
• Other Authors: Fu, Hua-Wen
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/59380608972706602279

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 98 === 英文摘要 Src, a member of Src non-receptor tyrosine kinase family, is the first protooncogene to be discovered and had been studied widely in tumorigenesis. In normal cells, Src is expressed ubiquitously and is involved in cell survival, proliferation, morphology, and motility. Contrary to normal cells, Src is overexpressed and highly activated in tumor cells. Therefore, it is important for cancer therapy to inhibit the expression and activation of Src in tumor cells. In the first part of this thesis, the recombinant Src was purified from E.coli expression system and was used to determine Km for ATP in Src autophosphorylation. It was found that the time course of Src autophosphorylation showed a sigmoidal curve. In addition, the two initiate rates, S1 and S2, were acquired by assuming that there were two catalytic reactions in the process of Src autophosphorylation. If the two reactions followed the Michaelis-Menten equation, the two values of Km for ATP of Src autophosphorylation were obtained and designated as Ks1 and Ks2. As a result, the Ks1 and Ks2 for ATP of the two catalytic reactions in the Src autophosphorylation were 23.8 μM and 64.7 μM, respectively. In the secondary part of this thesis, the ubiquitin-modified lysine residues of Src were predicted by bioinformatics analysis. Active Src could be down-regulated by ubiquitination. Furthermore, most of the ubiquitinated lysine residues are supposed to be exposed at the surface of the molecule. Therefore, the lysine residues were defined at the surface of Src molecule by calculating their relative accessible surface area in published structures of Src. To further search for the more probable residues for ubiquitination within these surface lysine residues, I took a look at the primary structure, secondary structure, and conservation analysis of amino acid sequences of these lysine residues. In conclusion, the most probable ubiquitination site of Src is Lys104. In addition, Lys423, Lys316, Lys298, Lys200, Lys203, and Lys272 also might be ubiquitin-modified residues in active Src.