人类激酶基因CK1γ1L2和PDIK1L的克隆和功能研究
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摘要
蛋白激酶是催化蛋白质磷酸化的酶,它们在细胞信号传导中发挥重要作用。蛋白激酶可以分为丝/苏氨酸蛋白激酶和酪氨酸蛋白激酶。酪蛋白激酶是广泛存在于真核生物的丝/苏氨酸蛋白激酶,包括CK1和CK2两大亚类。其中CK1是发现较早的,哺乳动物的CK1发现有α、β、γ、δ和ε等5个亚家族,CK1γ亚家族目前发现有CK1γ1、CK1γ2和 CK1γ3三个成员。还有一种既能催化丝/苏氨酸残基磷酸化又能催化酪氨酸残基磷酸化的激酶,这种具有双底物特异性的激酶称为双特异性蛋白激酶。
我们从胎脑cDNA文库中克隆得到两条人类的新激酶基因:CK1γ1L2和PDIK1L。CK1γ1L2为酪蛋白激酶CK1γ1的一个新的剪切体,该基因全长1756 bp,编码含438个氨基酸残基的蛋白,其核苷酸序列和氨基酸序列与大鼠CK1γ1基因相比分别为93%和94%的同源性。CK1γ1L2定位于人染色体15q22.31,包含11个外显子。亚细胞定位结果发现CK1γ1L2定位于细胞核中。Northern和Panel表达谱分析发现该基因分布于脑、肝、脾、外周血白细胞、肾、肺、小肠、卵巢、睾丸和前列腺中,在心和骨骼肌组织也可能有表达,另外CK1γ1L2在卵巢癌、结肠癌、前列腺癌、肺癌和乳腺癌中也检测到显著表达。
PDIK1L基因全长4301 bp,编码含有341个氨基酸残基的蛋白。通过生物信息学分析发现它的蛋白与丝/苏氨酸激酶和酪氨酸激酶催化功能域同源性较高,有可能是个双特异性激酶。PDIK1L与人激酶CLIK1蛋白同源性为69%,CLIK1是PDZ-LIM蛋白CLP-36的相关激酶,经HUGO基因命名委员会核准,将其命名为PDIK1L,即类PDLIM1相关激酶1(PDLIM1 interacting kinase 1 like)。该基因定位于人染色体1p36.11,包含4个外显子。亚细胞定位结果发现PDIK1L也定位在细胞核中。Panel表达谱分析发现该基因广泛表达于多种组织,只是不同区域的基因序列表达存在差异,除了在胰腺、前列腺、胸腺和脾中表达之外,PDIK1L基因中段第1781～2200区域还可在外周血白细胞、结肠、小肠、卵巢和睾丸中检测到表达,而3'端附近的第3296～3724区域可在肝、肾、心、脑和胎盘中检测到表达,这有可能是因为PDIK1L基因存在不同转录本所致,或许与该基因在生物体内的功能相关。激酶的活性测定发现PDIK1L蛋白具有丝/苏氨酸激酶活性。经酵母双杂交筛选人胎脑文库并经免疫共沉淀验证,发现PDIK1L能与GRINL1A(类离子型谷氨酸NMDA受体1A)相互作用。GRINL1A是近几年发现的谷氨酸受体家族的新成员。谷氨酸受体广泛分布于中枢神经系统,对于神经信号传递起着重要作用,PDIK1L与GRINL1A的相互作用为进一步揭示PDIK1L的功能提供了一定依据。
Protein kinases, which play an important role in cell signing transduction, is a kind of enzymes that can phosphorylate lots of proteins in the organism. There are two subvisions in the protein kinase superfamily, serine/threonine kinase and tyrosine kinase. Casein kinase are ubiquitous in eukaryotic organisms and yeasts, they belong to the serine/threonine kinase family and contain CK1 and CK2 subfamilies. Casein kinase I (CKI) was one of the first serine/threonine protein kinase activities to be isolated and characterised. Since then, mammalian CKI subfamilies designed α、β、γ、δ and ε have been cloned and characterized, γ1, γ2, γ3 are the members of CK1γ subfamily. Besides serine/threonine kinase and tyrosine kinase, there exist the dual-specificity protein kinases (DSPK), which possess both serine/threonine kinase and tyrosine kinase activity.
From a human fetal brain cDNA library, we isolated two novel human kinase genes, CK1γ1L2 and PDIK1L. CK1γ1L2 is a new isoform of CK1γ1. It contained 1756 bp and encoded a putative protein of 438 amino acids. The nucleotide and peptide sequences showed 93% and 94% identity with the rat CK1γ1. The gene CK1γ1L2 located in human chromosome 15q22.31 and contained 11 exons. The results of subcelluar localisation showed that CK1γ1L2 protein mainly located in the cell nucleus. The expression of CK1γ1L2 could be detected in brain, liver, spleen, peripheral blood leukocyte, kidney, lung, small intestine, ovary, testis and prostate, as well as heart and skeletal muscle according to the Northern Blot and RT-PCR analysis results. In addition, the results displayed that CK1γ1L2 expressed in ovarian carcinoma, colon carcinoma, prostatic adenocarcinoma, lung carcinoma and breast adenocarcinoma. It suggests CK1γ1L2 maybe a gene which associate with tumors.
PDIK1L contained 4301 bp and encoded a putative protein of 341 amino acids. Bioinformaic analysis represented that the 341-aa peptide showed over 90% homologous with both serine/threonine kinase and tyrosine kinase catalytic domain and 69% homologous with the human CLIK1, which act as the interacting kinase of the PDZ-LIM protein CLP-36. Therefore, approved by the HUGO Gene Nomenclature Committee, it was named PDIK1L, PDLIM1 interacting kinase 1 like. The gene PDIK1L located in human chromosome 1p36.11 and contained 4 exons. Results of subcelluar localisation showed that PDIK1L protein also located in the cell nucleus. According to the PT-PCR, we found PDIK1L expressed in various tissues such as pancreas, prostate, thymus and spleen. Expression signal of the mRNA sequence lies in the middle part (1781-2200) was detected in peripheral blood leukocyte, colon, small intestine, ovary and testis while expression signal of the
mRNA sequence close to the 3’end(3296-3724) was detected in liver, kidney, heart, brain and planceta, maybe the existence of different PDIK1L transcripts should responsible for the tissue-difference expression within the same gene. Kinase assay results demonstrate the serine/threonine kinase activity of PDIK1L. Two-yeast hybrid revealed that a potential glutamine receptor, GRINL1A, maybe associate with PDIKL1A, co-immunoprecipitation confirmed further the association between the two proteins. It may provied more proof and clue for the next research of the function of PDIK1L.
我们从胎脑cDNA文库中克隆得到两条人类的新激酶基因:CK1γ1L2和PDIK1L。CK1γ1L2为酪蛋白激酶CK1γ1的一个新的剪切体,该基因全长1756 bp,编码含438个氨基酸残基的蛋白,其核苷酸序列和氨基酸序列与大鼠CK1γ1基因相比分别为93%和94%的同源性。CK1γ1L2定位于人染色体15q22.31,包含11个外显子。亚细胞定位结果发现CK1γ1L2定位于细胞核中。Northern和Panel表达谱分析发现该基因分布于脑、肝、脾、外周血白细胞、肾、肺、小肠、卵巢、睾丸和前列腺中,在心和骨骼肌组织也可能有表达,另外CK1γ1L2在卵巢癌、结肠癌、前列腺癌、肺癌和乳腺癌中也检测到显著表达。
PDIK1L基因全长4301 bp,编码含有341个氨基酸残基的蛋白。通过生物信息学分析发现它的蛋白与丝/苏氨酸激酶和酪氨酸激酶催化功能域同源性较高,有可能是个双特异性激酶。PDIK1L与人激酶CLIK1蛋白同源性为69%,CLIK1是PDZ-LIM蛋白CLP-36的相关激酶,经HUGO基因命名委员会核准,将其命名为PDIK1L,即类PDLIM1相关激酶1(PDLIM1 interacting kinase 1 like)。该基因定位于人染色体1p36.11,包含4个外显子。亚细胞定位结果发现PDIK1L也定位在细胞核中。Panel表达谱分析发现该基因广泛表达于多种组织,只是不同区域的基因序列表达存在差异,除了在胰腺、前列腺、胸腺和脾中表达之外,PDIK1L基因中段第1781～2200区域还可在外周血白细胞、结肠、小肠、卵巢和睾丸中检测到表达,而3'端附近的第3296～3724区域可在肝、肾、心、脑和胎盘中检测到表达,这有可能是因为PDIK1L基因存在不同转录本所致,或许与该基因在生物体内的功能相关。激酶的活性测定发现PDIK1L蛋白具有丝/苏氨酸激酶活性。经酵母双杂交筛选人胎脑文库并经免疫共沉淀验证,发现PDIK1L能与GRINL1A(类离子型谷氨酸NMDA受体1A)相互作用。GRINL1A是近几年发现的谷氨酸受体家族的新成员。谷氨酸受体广泛分布于中枢神经系统,对于神经信号传递起着重要作用,PDIK1L与GRINL1A的相互作用为进一步揭示PDIK1L的功能提供了一定依据。
Protein kinases, which play an important role in cell signing transduction, is a kind of enzymes that can phosphorylate lots of proteins in the organism. There are two subvisions in the protein kinase superfamily, serine/threonine kinase and tyrosine kinase. Casein kinase are ubiquitous in eukaryotic organisms and yeasts, they belong to the serine/threonine kinase family and contain CK1 and CK2 subfamilies. Casein kinase I (CKI) was one of the first serine/threonine protein kinase activities to be isolated and characterised. Since then, mammalian CKI subfamilies designed α、β、γ、δ and ε have been cloned and characterized, γ1, γ2, γ3 are the members of CK1γ subfamily. Besides serine/threonine kinase and tyrosine kinase, there exist the dual-specificity protein kinases (DSPK), which possess both serine/threonine kinase and tyrosine kinase activity.
From a human fetal brain cDNA library, we isolated two novel human kinase genes, CK1γ1L2 and PDIK1L. CK1γ1L2 is a new isoform of CK1γ1. It contained 1756 bp and encoded a putative protein of 438 amino acids. The nucleotide and peptide sequences showed 93% and 94% identity with the rat CK1γ1. The gene CK1γ1L2 located in human chromosome 15q22.31 and contained 11 exons. The results of subcelluar localisation showed that CK1γ1L2 protein mainly located in the cell nucleus. The expression of CK1γ1L2 could be detected in brain, liver, spleen, peripheral blood leukocyte, kidney, lung, small intestine, ovary, testis and prostate, as well as heart and skeletal muscle according to the Northern Blot and RT-PCR analysis results. In addition, the results displayed that CK1γ1L2 expressed in ovarian carcinoma, colon carcinoma, prostatic adenocarcinoma, lung carcinoma and breast adenocarcinoma. It suggests CK1γ1L2 maybe a gene which associate with tumors.
PDIK1L contained 4301 bp and encoded a putative protein of 341 amino acids. Bioinformaic analysis represented that the 341-aa peptide showed over 90% homologous with both serine/threonine kinase and tyrosine kinase catalytic domain and 69% homologous with the human CLIK1, which act as the interacting kinase of the PDZ-LIM protein CLP-36. Therefore, approved by the HUGO Gene Nomenclature Committee, it was named PDIK1L, PDLIM1 interacting kinase 1 like. The gene PDIK1L located in human chromosome 1p36.11 and contained 4 exons. Results of subcelluar localisation showed that PDIK1L protein also located in the cell nucleus. According to the PT-PCR, we found PDIK1L expressed in various tissues such as pancreas, prostate, thymus and spleen. Expression signal of the mRNA sequence lies in the middle part (1781-2200) was detected in peripheral blood leukocyte, colon, small intestine, ovary and testis while expression signal of the
mRNA sequence close to the 3’end(3296-3724) was detected in liver, kidney, heart, brain and planceta, maybe the existence of different PDIK1L transcripts should responsible for the tissue-difference expression within the same gene. Kinase assay results demonstrate the serine/threonine kinase activity of PDIK1L. Two-yeast hybrid revealed that a potential glutamine receptor, GRINL1A, maybe associate with PDIKL1A, co-immunoprecipitation confirmed further the association between the two proteins. It may provied more proof and clue for the next research of the function of PDIK1L.
引文
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孙大业,郭艳林,马力耕。细胞信号转导。科学出版社,2000
杨新平,夏家辉 蛋白激酶研究进展 生命科学研究 1998 2(4):235-243
张龙翔 生化实验方法和技术 1981 北京 人民教育出版社 112-118