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

描述(申请人提供)：鉴定负责高尔基酪蛋白激酶活性的基因(S)和蛋白产物(S)。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)参与了S-X-E/PS基序上大量分泌蛋白的磷酸化，但该酶的基因来源尚不清楚。推测该酶对几种釉质细胞外基质(ECM)蛋白如釉原蛋白(AMEL)、釉蛋白(ENAM)和成釉蛋白(AMBN)的磷酸化起重要作用，这些蛋白都含有这个独特的SXE基序。这些蛋白质的突变，除了成釉蛋白，已经被证明会导致成釉不全(AI)，或釉质畸形。到目前为止，只有大约50%的已知AI病例有确认的遗传原因。这项研究可能证明GCK是AI的另一个致病基因，这将增加我们对这种遗传病和牙釉质形成的总体认识。到目前为止，GCK还只是生物化学的特征。因此，本研究的目的是确定与GCK相关的初级蛋白序列(S)和基因(S)。这项研究还将试图确定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活性定位于高尔基体细胞膜，并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)确定与GCK有关的主要蛋白质序列(S)和基因(S)；2)确定GCK活性是膜相关的还是一种含量的酶活性。这项研究计划使用大鼠肝脏匀浆，使用超速离心技术和可变蔗糖密度的溶液来纯化高尔基体组分。这些组分将进一步分离为高尔基膜部分和可溶性成分，将分别进行分析。高尔基体组分将用高效液相色谱进一步纯化，含有GCK的阳性组分将通过32P和只有GCK才能磷酸化的定制短肽的放射性标记分析来鉴定。高效液相色谱的活性峰将使用PF2D分馏系统进行进一步分馏，并将测试每个得到的级分的磷酸化能力。活性组份将接受2D-Gel分析，产生的斑点将被分离和测序。使用BLAST对序列进行检查，以确定不同物种之间相似的蛋白质和核苷酸序列，然后确定序列同源性百分比。将进行蛋白质功能域分析，并将使用GCK唯一序列来产生两个抗肽抗体。这些抗体将用于免疫组织化学，以确定GCK在肝脏切片中的表达模式。 与公共卫生相关：釉质发生不全(AI)是一种牙釉质畸形，发病率为1：700至14,000，可导致功能障碍和毁容。有证据表明，釉质细胞外基质蛋白的磷酸化对釉质的正常形成至关重要；在成釉细胞中调节这一事件的首要候选者是高尔基酪蛋白激酶。本项目的目的是分离和鉴定高尔基体酪蛋白激酶，以确定其活性的基因(S)和蛋白产物(S)，当受到干扰时，很可能导致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.