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

描述（由申请人提供）：鉴定负责高尔基酪蛋白激酶活性的基因和蛋白质产物。磷酸化是细胞调节多种功能的一种重要而普遍的方式。高尔基酪蛋白激酶（GCK）蛋白与S-X- E/pS基序上许多分泌蛋白的磷酸化有关，但目前尚不清楚该酶的起源基因。据推测，这种酶对几种釉质细胞外基质（ECM）蛋白的磷酸化很重要，如amelogenin （AMEL）、enamelin （ENAM）和ameloblastin (AMBN)，它们都含有这种独特的SXE基序。除成釉细胞蛋白外，这些蛋白的突变已被证明会导致成釉不完全性（AI）或牙釉质畸形。迄今为止，已知的人工智能病例中只有约50%有确定的遗传原因。这项研究可能会证明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认识。迄今为止，GCK只进行了生物化学表征。因此，本研究的目的是确定GCK的主要蛋白序列和基因。本研究还将尝试确定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活性定位于高尔基膜，并负责磷酸化淀粉原蛋白。为了研究这一假设，研究有两个目的：1)确定负责GCK的主要蛋白质序列和基因；2)确定GCK活性是与膜相关还是与含量酶活性相关。本研究计划采用超离心技术和可变蔗糖密度溶液，利用大鼠肝脏匀浆纯化高尔基体。这些组分将进一步分离成高尔基膜部分和可溶性组分，分别进行分析。高尔基体馏分将使用高效液相色谱（HPLC）进一步纯化，含有GCK的阳性馏分将使用32P和只有GCK才能磷酸化的短定制肽进行放射性标记测定。高效液相色谱的活性峰将使用PF2D分馏系统进一步分馏，每个分馏部分将被测试磷酸化能力。活性组分将进行2d凝胶分析，得到的斑点将被分离和测序。利用BLAST对序列进行比对，找出不同物种间相似的蛋白和核苷酸序列，确定序列同源率。进行蛋白功能域分析，利用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来确定肝切片中GCK的表达模式。

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. PUBLIC HEALTH RELEVANCE: Amelogenesis imperfecta (AI) with an incidence of 1:700 to 14,000 is a malformation of the tooth enamel, which can be dysfunctional and disfiguring. Evidence supports that phosphorylation of enamel extracellular matrix proteins is critical for proper enamel formation; the prime candidate in regulating such event in ameloblast is Golgi Casein Kinase. The purpose of this project is to isolate and characterize Golgi Casein Kinase to identify its gene(s) and protein product(s) responsible for its activities, which when disturbed is likely to result in AI.