中文摘要

非受器型Src酪胺酸激酶家族成員之一的Src酪胺酸激酶是第一個被發現的原致癌基因，常被用於癌症相關的研究。Src酪胺酸激酶廣泛地存在於細胞中且參與正常細胞的存活、生長、細胞型態、及移動。然而，Src酪胺酸激酶常以過度表現及高度活化的情況存在於腫瘤細胞中。因此，抑制Src酪胺酸激酶在腫瘤細胞的活性或降低其蛋白質的表現量對於癌症的治療是極為重要的。本文第一部份中，主要是從大腸桿菌中純化出重組Src酪胺酸激酶並探討其酵素進行自體磷酸化對於ATP的Km值。實驗發現，Src酪胺酸激酶自體磷酸化作用與時間關係圖呈現S型曲線 (sigmoidal curve)。除此之外，在Src酪胺酸激酶自體磷酸化是由兩個催化反應構成的推論中，可獲得兩個初速度S1及S2。假設這兩個反應皆符合Michaelis-Menten公式的條件，則Src酪胺酸激酶自體磷酸化對於ATP將會有兩個Km值且於此處定為Ks1及Ks2。由結果可知Src酪胺酸激酶自體磷酸化中的兩個催化反應對於ATP的Ks1及Ks2值分別為23.8 μM及64.7 μM。本文第二部份主要藉由生物資訊分析法來探討Src酪胺酸激酶上泛素化的離胺酸殘基。已知活化態的Src酪胺酸激酶才可進行泛素化的作用進而導致降解作用。除此之外，可被泛素修飾的離胺酸殘基大多位在蛋白質分子表面。因此，藉由計算已發表的Src酪胺酸激酶結構上離胺酸殘基與溶劑可接觸的表面積 (accessible surface area，簡稱 ASA值)，找出暴露於此酵素分子表面的離胺酸殘基。為了更進一步地探討可被泛素修飾的離胺酸殘基，再針對離胺酸殘基所在的一級結構、二級結構以及離胺酸殘基保留性這三個方面去深入探討。結果發現Src酪胺酸激酶最有可能泛素化的位置為離胺酸殘基序號104。此外，離胺酸殘基序號423、316、298、200、203及272也有可能是Src酪胺酸激酶上泛素化的位置。

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.

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