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.
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.
