Functions of Family with Sequence Similarity 20 - Member C (FAM20C) and Member A (FAM20A) in Amelogenesis and Dentinogenesis

项目总结 磷酸化是磷酸化蛋白质发挥其调节相关功能的关键。 生物过程。最近的研究表明，一种被称为FAM20C(家族成员)的蛋白激酶 序列相似性20-成员C)与其并列对数(命名为“FAM20A”)一起发挥关键和非冗余作用 分泌蛋白的Ser-x-Glu/pSer基序中丝氨酸残基的磷酸化功能，包括 牙釉质和牙本质基质蛋白是釉质形成和牙本质形成所必需的。尽管研究表明 已确认FAM20C驻留在高尔基仪器中，目前尚不清楚FAM20C是如何 一个跨膜区，保留在这个细胞器中，以发挥其激酶功能。而体外研究表明 FAM20A可以增强FAM20C的激酶活性，无论这种功能相互作用是否发生在 体内成釉细胞和成牙本质细胞仍有待确定；人类FAM20A突变或其失活 在小鼠中只影响牙釉质的形成，而对牙本质的形成没有病理影响，而 FAM20C功能导致牙釉质和牙本质缺陷。该项目的目标是确定1) 控制FAM20C高尔基体滞留的分子机制，以及2)FAM20C与FAM20A的相互作用 在釉质形成和牙本质形成过程中。初步研究表明，1)与FAM20C相关 当FAM20C存在时，细胞提取液中存在超分子复合体，而培养基中没有超分子复合体 2)在转基因细胞中也发现了一条与FAM20C相似的大蛋白条带 从釉质器官(含成釉细胞)和牙髓(含成牙本质细胞)中提取的蛋白质 3)FAM20a基因缺陷的小鼠出现釉质缺陷，但没有牙本质异常，而 FAM20c基因缺陷的小鼠有牙釉质和牙本质的缺陷；4)FAM20a基因缺陷的小鼠的牙本质缺陷显著减少 在编码釉质基质蛋白的基因的表达水平上，但在 编码主要牙本质基质蛋白的基因；5)FAM20A缺失降低了FAM20C的水平 成釉细胞和成牙本质细胞中的蛋白质。这些发现导致假设FAM20C形成一个 超分子复合体，通过它将该激酶保留在高尔基体内，并且 FAM20A拓宽了FAM20C的底物谱，是正确磷酸化FAM20C所必需的 疏水性釉质基质蛋白。提出了两个目标来检验这一新的假设：目标1-到 确定FAM20C在成釉细胞和成牙本质细胞中高尔基体滞留的机制。目标2 -确定FAM20A和FAM20C在成釉细胞和成牙本质细胞中的功能。成功完成 将不仅阐明FAM20C和FAM20A发挥作用的分子机制 在釉质和牙本质基质蛋白的磷酸化中，但也可能为发展的线索 FAM20C和FAM20A突变引起的疾病的临床治疗策略。

PROJECT SUMMARY Phosphorylation is essential for the phosphorylated proteins to exert their functions in regulating the relevant biological processes. Recent studies have demonstrated that a protein kinase known as “FAM20C” (Family with sequence similarity 20 – member C), together with its paralog named “FAM20A”, plays key and non-redundant functions in phosphorylating serine residues within the Ser-x-Glu/pSer motifs of the secretory proteins, including enamel and dentin matrix proteins that are essential to amelogenesis and dentinogenesis. Although studies have confirmed that FAM20C resides in the Golgi apparatus, it is unclear as to how FAM20C, which does not possess a transmembrane domain, is retained in this organelle to exert its kinase function. While in vitro studies suggest that FAM20A may enhance the kinase activity of FAM20C, whether such functional interaction occurs in ameloblasts and odontoblasts in vivo remains to be determined; FAM20A mutations in humans or its inactivation in mice only affect enamel formation but have no pathological effect on dentin formation, whereas loss of FAM20C function leads to both enamel and dentin defects. The goals of this project are to determine 1) the molecular mechanisms that govern the Golgi-retention of FAM20C, and 2) how FAM20C interacts with FAM20A during amelogenesis and dentinogenesis. Preliminary studies have shown that 1) a FAM20C-related supramolecular complex was present in the cell extract but not in the culture medium when FAM20C was expressed in the transfected cells; 2) a similar large FAM20C-related protein band was also found in the total proteins extracted from the enamel organ (containing ameloblasts) and dental pulp (containing odontoblasts) of mouse teeth; 3) Fam20a-deficient mice developed enamel defects, but had no dentin abnormalities, whereas Fam20c-deficient mice had both enamel and dentin defects; 4) Fam20a-deficient mice had a dramatic reduction in the expression levels of genes encoding the enamel matrix proteins, but showed no expression changes in the genes encoding the major dentin matrix proteins; and 5) Fam20a deletion reduced the level of FAM20C protein in ameloblasts and odontoblasts. These findings lead to the hypothesis that FAM20C forms a supramolecular complex, through which this kinase is retained within the Golgi apparatus, and that FAM20A broadens the substrate spectrum of FAM20C and is required for the proper phosphorylation of the hydrophobic enamel matrix proteins. Two Aims are proposed to test this novel hypothesis: Aim 1 – to determine the mechanisms that govern the Golgi-retention of FAM20C in ameloblasts and odontoblasts. Aim 2 – to determine the functions of FAM20A and FAM20C in ameloblasts and odontoblasts. Successful completion of the proposed work will not only elucidate the molecular mechanisms by which FAM20C and FAM20A function in the phosphorylation of enamel and dentin matrix proteins, but may also provide clues for the development of therapeutic strategies for the clinical management of diseases caused by the FAM20C and FAM20A mutations.