Phosphorylation of FGF23 coordinates crosstalk between the skeleton and kidney

描述（由申请人提供）：研究：蛋白激酶的蛋白磷酸化是影响细胞生命各个方面的重要调控机制。大多数磷酸化蛋白在细胞内；然而，许多细胞外蛋白也被磷酸化。事实上，蛋白质磷酸化的第一个证据是在1883年，当时分泌的牛奶蛋白酪蛋白被证明含有磷酸盐。事后看来，这是蛋白激酶存在的第一个迹象。许多分泌的蛋白质被存在于高尔基体管腔内的蛋白激酶磷酸化。然而，这些酶的特性很差，它们的活性被归为孤儿酶（未被分子鉴定的酶）。高尔基酪蛋白激酶（Golgi casein kinase, G-CK）是一种从高尔基蛋白中分离出来的激酶。被称为“酪蛋白激酶”的蛋白质实际上是细胞质蛋白和核蛋白，它们不介导酪蛋白的生理磷酸化，因为它们在空间上受到分泌器官和细胞外空间的限制。G-CK特异性识别一致的Ser-x-(Glu/pSer)（其中x是任何氨基酸，Glu/pSer可以是Glu或phospho-Ser），该基序在大约75%的人血浆和脑脊液磷酸化蛋白中被磷酸化。候选人是第一个确定G-CK为Fam20C的人，并表明它属于一个新的非典型蛋白激酶家族，该家族位于高尔基体内并分泌。Fam20C磷酸化酪蛋白和高尔基体管腔S-x-E/pS基序上的几种激素。事实上，许多激素在这个基序中含有磷酸盐；然而，在大多数情况下，确定的磷酸化功能是未知的。在候选物发现Fam20C是一种蛋白激酶之前，它被认为是成纤维细胞生长因子23 （FGF23）的一种新型调节剂。FGF23是一种骨源性激素，与许多人类疾病的发病机制有关，包括矿物质代谢紊乱和慢性肾脏疾病。假设：本提案旨在验证分泌的蛋白激酶Fam20C通过磷酸化调节FGF23的假设。该假设是基于最近的三个观察得出的：1)Fam20C是一种磷酸化S-x-E/pS分泌蛋白的蛋白激酶（Tagliabracci et. al., Science 2012）。2)由于FGF23的增加，Fam20C缺失小鼠会发生低磷性佝偻病（Wang等人，PLoS Genet. 2012）。3)Fam20C使FGF23在一个重要调控基元的180号丝氨酸磷酸化，并在C端3个高度保守的氨基酸磷酸化，这可能会影响激素与其同源受体的结合（初步数据，未发表）。研究目的：本提案的总体目标是确定Fam20C调控FGF23加工和活性的分子机制。方法：候选人将利用分子生物学、生物化学、细胞生物学和小鼠遗传学的结合来完成研究目标。指导阶段（K99， Specific Aims 1和2）将包括体外和基于细胞的方法，以确定Fam20C调节FGF23的分子机制。候选人将利用加州大学圣地亚哥分校的资源和人员，这两者都是优秀的，非常适合该奖项的这个阶段。独立阶段（Specific Aim 3）将采用小鼠模型研究Fam20C对FGF23的调控作用。意义：本应用程序中提出的实验将回答有关几种矿物质代谢紊乱的发病机制的基本问题，包括遗传性低磷血症佝偻病；每2万名新生儿中约有1人受影响。此外，研究结果将对慢性肾病患者产生重要影响。慢性肾病是一种日益严重的公共卫生流行病，影响着2600万美国人。培训：拟议的研究将在加州大学圣地亚哥分校药学系进行，在Jack E. Dixon博士的指导下进行。Dixon博士是美国国家科学院（National Academy of Sciences， USA）的成员、皇家学会（Royal Society）会员，并领导了杰出的科学生涯。此外，还与代谢性骨病领域的领导者Kenneth White博士和著名肾病学家和流行病学家Joachim H. Ix医学博士建立了合作关系。候选人将参加加州大学圣地亚哥分校的课程，参加定期研讨会，并在国家科学会议上展示他的发现。总的来说,

DESCRIPTION (provided by applicant): Research: Protein phosphorylation by protein kinases is an important regulatory mechanism that influences every aspect of cellular life. The majority of phosphoproteins are intracellular; however, numerous extracellular proteins are also phosphorylated. In fact, the first evidence of protein phosphorylation was in 1883, when the secreted milk protein casein was shown to contain phosphate. In hindsight, this was the first indication for the existence of protein kinases. Many secreted proteins are phosphorylated by protein kinases present within the lumen of the Golgi. However, these enzymes are poorly characterized and their activities have been ascribed to the category of orphan enzymes (enzymes that have not been molecularly identified). One such kinase has been biochemically characterized from highly enriched Golgi fractions and named Golgi casein kinase (G-CK). The proteins known as "casein kinases" are in fact cytosolic and nuclear proteins and do not mediate physiological phosphorylation of casein because they are spatially restricted from the secretory apparatus and the extracellular space. The G-CK specifically recognizes the consensus Ser-x-(Glu/pSer) (where x is any amino acid and Glu/pSer can be Glu or phospho-Ser) and this motif is phosphorylated in some 75% of human plasma and cerebrospinal fluid phosphoproteins. The candidate was the first to identify the G-CK as Fam20C, and showed that it belongs to a novel family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C phosphorylates casein and several hormones specifically at S-x-E/pS motifs in the lumen of the Golgi. Indeed, many hormones contain phosphate within this motif; however, in most cases, established functions for phosphorylation are unknown. Prior to the candidates' discovery that Fam20C was a protein kinase, it had been suggested to be a novel regulator of Fibroblast Growth Factor 23 (FGF23). Fam20C null mice develop hypophosphatemic rickets (HR) as a result of elevated secretion of FGF23, a bone- derived hormone implicated in the pathogenesis of many human diseases including disorders of mineral metabolism and chronic kidney disease. Hypothesis: The aims of this proposal were designed to test the hypothesis that the secreted protein kinase Fam20C regulates FGF23 by phosphorylation. The hypothesis was formulated based on three recent observations: 1) Fam20C is a protein kinase that phosphorylates secreted proteins at S-x-E/pS (Tagliabracci et. al., Science 2012). 2) Fam20C null mice develop hypophosphatemic rickets due to an increase in FGF23 (Wang et. al., PLoS Genet. 2012) and 3) Fam20C phosphorylates FGF23 at Ser180 within an important regulatory motif and at 3 highly conserved amino acids within the C terminus that may affect binding of the hormone to its cognate receptor (preliminary data, unpublished). Research Objective: The overall objective of this proposal is to determine the molecular mechanisms by which Fam20C regulates FGF23 processing and activity. Approach: The candidate will utilize a combination of molecular biology, biochemistry, cell biology, and mouse genetics to accomplish the research objective. The mentored phase (K99, Specific Aims 1 and 2) will consist of in vitro and cell based approaches to determine the molecular mechanism by which Fam20C regulates FGF23. The candidate will take advantage of the resources and personnel at UCSD, both of which are outstanding and well-suited for this phase of the award. The independent phase (Specific Aim 3) will employ mouse models to study the regulation of FGF23 by Fam20C. Significance: The experiments proposed in this application will answer fundamental questions regarding the pathogenesis of several disorders of mineral metabolism, including hereditary disorders of hypophosphatemic rickets; affecting ~1 in every 20,000 newborns. Furthermore, the results will have important implications for patients with chronic kidney disease, a growing public health epidemic that affects 26 million Americans. Training: The proposed research will be conducted within the department of Pharmacology at the University of California, San Diego (UCSD), under the mentorship of Dr. Jack E. Dixon. Dr. Dixon is a member of the National Academy of Sciences, USA, a Royal Society Fellow, and has led a distinguished scientific career. In addition, collaborations were established with Kenneth White, PhD, a leader in the field of metabolic bone diseases and Joachim H. Ix, MD, a noted nephrologist and epidemiologist. The candidate will undertake coursework through UCSD, participate in regular seminars, and present his findings at national scientific meetings. Overall, the training environment is excellent and the proposed studies are well designed and innovative. The hypothesis addresses a significant question that should yield influential findings in endocrinology, nephrology cardiology and basic biomedical research. The mentoring and research skills expected to develop over the course of the award will provide a strong foundation for the candidate to successfully transition to become a prominent independent investigator and leader in the field of secreted protein phosphorylation.