Identification of the gene and protein product responsible for Golgi casein kinas

鉴定负责高尔基酪蛋白激酶的基因和蛋白质产物

• 批准号: 8774877
• 负责人: Stephanie Marie Nunez
• 金额: $ 2.23万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8774877
• 关键词: Affect; Ameloblasts; Amelogenesis Imperfecta; Amino Acid Sequence; Antibodies; Base Sequence; Biological Assay; CSNK1A1 gene; Calcium Binding; Caseins; Cattle; Cells; Cleaved cell; Custom; Dental Enamel; Detergents; Enamel Formation; Enzymes; Event; Extracellular Matrix Proteins; Fractionation; Gel; Gene Proteins; Genes; Genetic; Golgi Apparatus; Hereditary Disease; High Pressure Liquid Chromatography; Hydrophobicity; Immunohistochemistry; Incidence; Integral Membrane Protein; Label; Length; Liquid substance; Liver; Membrane; Membrane Proteins; Methods; Milk; Mutation; Pattern; Peptide Fragments; Peptide Sequence Determination; Peptide antibodies; Peptides; Peripheral; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein Family; Protein Kinase; Protein Kinase Inhibitors; Proteins; Radiolabeled; Rattus; Regulation; Resistance; Role; Sampling; Scintillation Counter; Series; Side; Sodium Chloride; Solutions; Speed; Spottings; Staurosporine; Sucrose; System; Techniques; Testing; Transmission Electron Microscopy; amelogenin; base; casein kinase; casein kinase II; density; enamelin; enzyme activity; luminal membrane; malformation; member; protein kinase inhibitor; radiotracer

DESCRIPTION (provided by applicant): Identification of the gene(s) and protein product(s) responsible for Golgi casein kinase activity. Phosphorylation is an important and ubiquitous way for cells to regulate a multitude of functions. The protein Golgi casein kinase (GCK) has been implicated in the phosphorylation of numerous secreted proteins at S-X- E/pS motifs but yet, the gene or genes that give origin to this enzyme is(are) not known. It is speculated that this enzyme is important for the phosphorylation of several enamel extracellular matrix (ECM) proteins such as amelogenin (AMEL), enamelin (ENAM), and ameloblastin (AMBN) which all contain this unique SXE motif. Mutations in these proteins, with the exception of ameloblastin, have been shown to cause amelogenesis imperfecta (AI), or malformation of the enamel. To date only about 50% of the known AI cases have a confirmed genetic cause. This study may demonstrate that GCK is another causative gene of AI, which would increase our understanding of this genetic disease and the formation of dental enamel in general. To date, GCK has only been characterized biochemically. Therefore, the purpose of this study is to identify the primary protein sequence(s) and gene(s) responsible for GCK. This study will also try to determine whether GCK activity preferentially correlates with Golgi membrane or its soluble components as this will affect how the protein could function. Therefore, I hypothesized that GCK activity is localized to the Golgi membrane and that it is responsible for phosphorylating amelogenin. To study this hypothesis the study has two aims: 1) to identify the primary protein sequence(s) and gene(s) responsible for GCK and 2) to determine if GCK activity is membrane associated or a content enzyme activity. This study plans to use rat liver homogenates to purify Golgi fractions using an ultracentrifuge technique and solutions of variable sucrose density. These fractions will be further separated into the Golgi membrane portion and soluble components, which will be analyzed separately. The Golgi fractions will be further purified using high performance liquid chromatography (HPLC) and positive fractions containing GCK will be identified using a radiolabeling assay with 32P and short custom peptides that only GCK can phosphorylate. Active peaks from HPLC will be further fractionated using a PF2D fractionation system and each resulting fraction will be tested phosphorylative capability. Active fractions will be subjected to 2D-gel analysis and resulting spots will be isolated and sequenced. Sequences will be checked using BLAST to identify similar protein and nucleotide sequences among various species, and then the percent sequence identity will be determined. Protein functional domain analyses will be conducted and the GCK unique sequence will be used to generate two anti-peptide antibodies. These antibodies will be used for immunohistochemistry to determine GCK expression patterns in liver sections.

描述（由申请人提供）：负责高尔基体酪蛋白激酶活性的基因和蛋白质产物的鉴定。磷酸化是细胞调节多种功能的重要且普遍的方式。高尔基体酪蛋白激酶 (GCK) 与许多分泌蛋白在 S-X-E/pS 基序上的磷酸化有关，但产生该酶的一个或多个基因尚不清楚。据推测，这种酶对于几种釉质细胞外基质 (ECM) 蛋白的磷酸化非常重要，例如釉原蛋白 (AMEL)、釉质蛋白 (ENAM) 和成釉素 (AMBN)，这些蛋白都含有这种独特的 SXE 基序。除成釉细胞外，这些蛋白质的突变已被证明会导致釉质形成不全（AI）或牙釉质畸形。迄今为止，已知的 AI 病例中只有约 50% 有明确的遗传原因。这项研究可能表明 GCK 是 AI 的另一个致病基因，这将增加我们对这种遗传疾病和牙釉质形成的总体了解。迄今为止，GCK 仅得到了生化特征。因此，本研究的目的是确定负责 GCK 的主要蛋白质序列和基因。这项研究还将尝试确定 GCK 活性是否优先与高尔基膜或其可溶性成分相关，因为这将影响蛋白质的功能。因此，我假设 GCK 活性位于高尔基膜，并且负责磷酸化牙釉蛋白。为了研究这一假设，本研究有两个目的：1) 鉴定负责 GCK 的主要蛋白质序列和基因；2) 确定 GCK 活性是否与膜相关或内容酶活性。本研究计划使用大鼠肝脏匀浆，通过超速离心技术和不同蔗糖密度的溶液来纯化高尔基体级分。这些部分将进一步分离成高尔基膜部分和可溶性成分，并分别进行分析。高尔基体级分将使用高效液相色谱 (HPLC) 进一步纯化，含有 GCK 的阳性级分将使用放射性标记测定法进行鉴定，其中使用 32P 和只有 GCK 可以磷酸化的短定制肽。来自 HPLC 的活性峰将使用 PF2D 分级系统进一步分级，并且将测试所得的每个级分的磷酸化能力。活性组分将进行 2D 凝胶分析，所得斑点将被分离和测序。将使用 BLAST 检查序列，以识别不同物种之间相似的蛋白质和核苷酸序列，然后确定序列同一性百分比。将进行蛋白质功能域分析，并使用 GCK 独特序列生成两种抗肽抗体。这些抗体将用于免疫组织化学测定肝切片中的 GCK 表达模式。