丝氨酸/苏氨酸蛋白质激酶BRSK2功能的初步研究

【作者】 郭泽坤；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2004， 博士

【摘要】 蛋白激酶通过对蛋白底物的丝氨酸、苏氨酸或酪氨酸残基的磷酸化调节蛋白质的生物活性状态，这一过程在许多细胞活动中起着十分重要的作用，例如，细胞内外信号传导，细胞内运输，细胞周期进程等。我们通过电子杂交的方法从Genbank数据库中获得了BRSK2基因序列，该基因含有2025bp的开放阅读框，编码674个氨基酸残基，定位于11q15.5，含有21个外显子。BRSK2的氨基酸序列19-270位氨基酸残基编码一个Ser／Thr蛋白质激酶结构域，根据其激酶结构域的氨基酸序列同源性，将其归类于CAMK蛋白激酶家族的AMPK亚家族。BRSK2氨基酸序列的10-674位氨基酸残基还含有一个与细胞周期调控、细胞分裂以及染色体分离相关的KOG0588结构域，而且BRSK2是AMPK家族成员中唯一具有完整该结构域的蛋白激酶。组织表达谱分析表明BRSK2在脑和胰隙中呈现特异性的高表达，并且胰腺组织中的表达丰度要比脑组织中的表达丰度高10倍以上。免疫荧光实验将BRSK2在HeLa细胞内定位于核周区域。根据BRSK2激酶结构域与AMPK的同源性，我们用AMPK的底物SAMS多肽来检测BRSK2的激酶活性，实验结果表明BRSK2能够磷酸化SAMS多肽，而不能磷酸化突变多肽SAMA。对BRSK2细胞功能的研究发现BRSK2在葡萄糖饥饿胁迫条件下能够促进细胞存活率，抑制蛋白质合成，阻滞细胞周期在G2／M期，以及激活AP1、NFAT、HSE等细胞信号转导通路。我们在BRSK2发现了一个PKA的磷酸化位点Thr260，实验证明Thr260能够在体外被PKA磷酸化，并且PKA的磷酸化能够促进BRSK2的活性，将Thr260突变为丙氨酸，可导致激酶活性的降低，并导致BRSK2在促进细胞存活、蛋白质合成抑制、G2／M期阻滞和AP1激活等方面的功能的部分降低。已经有报道证明LKB1能够磷酸化激活BRSK2的Thr174，Thr174的突变可导致BRSK2活性的丧失。我们对比了Thr174以及Thr260位点的突变对BRSK2细胞功能的影响。实验结果表明Thr174位点的突变导致BRSK2功能的完全丧失，而Thr260的突变仅导致BRSK2功能的部分丧失，说明Thr174位点的磷酸化是BRSK2活性和功能所必需，该位点的突变将导致BRSK2功能的完全丧失，而Thr260的磷酸化对激酶活性具有调控作用，可能调控Thr174位点的磷酸化。更多还原

【Abstract】 Protein kinases achieve post-translational modifications via the phosphorylation of serine, threonine, and tyrosine residues, which plays a role in many cellular processes such as transduction of extracellular signals, intracellular transport, and cell cvcle Droeression.cell cycle progression.We obtained the gene sequence of BRSK2 from Genbank database by electric hybridization. This gene contains an open reading frame of 2025bp, encoding 674 amino acids, mapped to human chromosome 11q15.5 and composed of 21 exons. The 19-270 amino acid residues of amino acid of BRSK2 consist of a serine/threonine protein kinase domain. According to its amino acids sequence homology of kinase domain, it is classified to AMPK subfamily of CAMK protein kinase family. The 10-674 amino acid residues of amino acid of BRSK2 consist of a KOG0588 domain related to control of cell cycle, cell division and chromosome separation. Northern analysis indicates that BRSK2 highly expresses in brain and pancreas specifically. Subcellular localization of BRSK2 is around the nucleus.Based on the homology of BRSK2 to AMPK, the substrate polypeptide of AMPK, SAMS peptide, was performed to detect the kinase activity of BRSK2. The result shows that BRSK2 phosphorelates SAMS peptide but not the mutated SAMA peptide. Further study of BRSK2 indicates that BRSK2 possesses comprehensive cell function, such as increasing the cell survival under glucose starvation, suppressing the protein synthesis, causing the cell cycle G2/M delay, activating AP1, NFAT, HSE cell signal pathways.We identified a phosphorelation site of PKA at Thr260. In vitro experiments indicate that Thr260 can be phosphorelated by PKA. The phosphorelation at Thr260 canstimulate the activity of BRSK2. When Thr260 was mutated to Alanine, The kinase activity would depress, which would cause partial loss of function of BRSK2 on stimulating cell survival, control of protein synthesis, G2/m stage and AP1 activation.It has been reported that LKB1 can phosphorelate and activate BRSK2 at Thr174 site, whose mutation prevented activation of BRSK2. The effect of Thr174 mutation on the cellular function of BRSK2 was compared to that of Thr260 mutation. The results indicate that the Thrl74 mutation causes the complete loss of function of BRSK2, whereas Thr260 mutation causes the partial loss of function. These results suggest that the phosphorelation at Thr174 is essential for activity and function of BRSK2, while phosphorelation at Thr260 modulates the kinase activity and the phosphorelation at Thr174.更多还原