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

描述（由申请方提供）：负责高尔基体酪蛋白激酶活性的基因和蛋白产物的鉴定。磷酸化是细胞调节多种功能的重要且普遍存在的方式。蛋白质高尔基体酪蛋白激酶（GCK）已经涉及在S-X- E/pS基序处的许多分泌蛋白质的磷酸化，但是还不知道产生该酶的一个或多个基因。据推测，这种酶是重要的几个釉细胞外基质（ECM）蛋白，如釉原蛋白（AMEL），釉蛋白（ENAM），成釉蛋白（AMBN），都含有这个独特的SXE基序的磷酸化。除成釉蛋白外，这些蛋白质的突变已被证明会导致釉质发生异常（AI）或釉质畸形。到目前为止，只有大约50%的已知AI病例具有确认的遗传原因。本研究可能证明GCK是AI的另一个致病基因，这将增加我们对这种遗传性疾病和牙釉质形成的认识。到目前为止，GCK仅进行了生物化学表征。因此，本研究的目的是鉴定负责GCK的主要蛋白质序列和基因。本研究还将试图确定GCK活性是否优先与高尔基体膜或其可溶性组分相关，因为这将影响蛋白质的功能。因此，我假设GCK活性定位于高尔基体膜，并且它负责磷酸化釉原蛋白。为了研究这一假说，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活性是膜相关的还是内容物酶活性。本研究计划使用大鼠肝匀浆纯化高尔基体组分，使用超离心技术和可变蔗糖密度的解决方案。这些组分将进一步分离成高尔基体膜部分和可溶性组分，分别进行分析。将使用高效液相色谱法（HPLC）进一步纯化高尔基体组分，并使用放射性标记试验（使用32 P和仅GCK可磷酸化的短定制肽）鉴定含有GCK的阳性组分。将使用PF 2D分馏系统进一步分馏HPLC中的活性峰，并检测所得各馏分的磷酸化能力。活性组分将进行2D凝胶分析，所得斑点将分离并测序。将使用BLAST检查序列，以鉴定不同种属之间的相似蛋白质和核苷酸序列，然后确定序列同一性百分比。将进行蛋白质功能结构域分析，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，以确定肝脏切片中的GCK表达模式。 公共卫生相关性：牙釉质发育异常（AI）是一种牙釉质畸形，其发病率为1：700至14，000，可能功能障碍和毁容。有证据表明，釉质细胞外基质蛋白的磷酸化对于正常的釉质形成是至关重要的;在成釉细胞中调节这种事件的主要候选者是高尔基体酪蛋白激酶。本项目的目的是分离和表征高尔基体酪蛋白激酶，以确定其基因和蛋白质产物，负责其活动，当受到干扰时可能导致AI。

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.