Phosphorylation of FGF23 coordinates crosstalk between the skeleton and kidney

FGF23 的磷酸化协调骨骼和肾脏之间的串扰

• 批准号: 8565659
• 负责人: Vincent Scott Tagliabracci
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8565659
• 关键词: Accounting; Address; Affect; American; Amino Acids; Award; Binding; Biochemistry; Biomedical Research; Blood Circulation; C-terminal; Calcinosis; California; Cardiology; Cardiovascular system; Caseins; Categories; Cell Nucleus; Cells; Cellular biology; Cerebrospinal Fluid; Chronic Kidney Failure; Collaborations; Consensus; Cytosol; Data; Disease; Doctor of Philosophy; Drosophila pros protein; Endocrinology; Environment; Enzymes; Epidemic; Epidemiologist; Eukaryotic Cell; Event; Extracellular Protein; Extracellular Space; Familial hypophosphatemic bone disease; Family; Fibroblast Growth Factor Receptors; Foundations; Gene Family; Genetic; Genets; Goals; Golgi Apparatus; Hereditary Disease; Homeostasis; Hormone Receptor; Hormones; Human; Human Resources; In Vitro; Influentials; Kidney; Knockout Mice; Length; Life; Mediating; Medicare; Mentors; Mentorship; Metabolic Bone Diseases; Metabolism; Milk Proteins; Minerals; Modeling; Molecular; Molecular Biology; Mus; Names; Nephrology; Newborn Infant; Nuclear Proteins; Orphan; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Pharmacology; Phase; Phosphoproteins; Phosphorylation; Phosphotransferases; Physiological; Plasma; Polypeptide N-acetylgalactosaminyltransferase; Process; Protein Kinase; Protein Kinase Protein Phosphorylation; Proteins; Proteolysis; Proteolytic Processing; Public Health; Receptor Activation; Regulation; Research; Research Personnel; Resources; Science; Serum; Site; Skeleton; Societies; Subtilisins; Testing; Therapeutic; Training; United States National Academy of Sciences; Universities; Work; absorption; base; bone; bone cell; career; casein kinase; design; fibroblast growth factor 23; glycosylation; human disease; in vivo; in vivo Model; innovation; inorganic phosphate; meetings; member; mortality; mouse model; novel; public health relevance; receptor; research study; skills

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.

描述（由申请人提供）：研究：蛋白激酶引起的蛋白磷酸化是影响细胞生命各个方面的重要调节机制。大多数磷蛋白是细胞内的;然而，许多细胞外蛋白也被磷酸化。事实上，蛋白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内腔中的蛋白激酶磷酸化。然而，这些酶的特性很差，它们的活性被归为孤儿酶（尚未被分子鉴定的酶）。一种这样的激酶已经从高度富集的高尔基体组分中进行了生物化学表征，并命名为高尔基体酪蛋白激酶（G-CK）。被称为“酪蛋白激酶”的蛋白质实际上是胞质和核蛋白，并且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和细胞外空间的限制。G-CK特异性识别共有Ser-x-（Glu/pSer）（其中x是任何氨基酸，Glu/pSer可以是Glu或磷酸-Ser），该基序在约75%的人血浆和脑脊液磷蛋白中被磷酸化。该候选人是第一个将G-CK鉴定为Fam 20 C的人，并表明它属于一个新的非典型蛋白激酶家族，该家族定位于高尔基体内并分泌。Fam 20 C磷酸化酪蛋白和几种激素，特别是在高尔基体腔中的S-x-E/pS基序。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，磷酸化的既定功能是未知的。在候选人发现Fam 20 C是一种蛋白激酶之前，它被认为是成纤维细胞生长因子23（FGF 23）的一种新的调节剂。Fam 20 C缺失型小鼠由于FGF 23分泌升高而发生低磷酸盐血症性佝偻病（HR），FGF 23是一种与许多人类疾病（包括矿物质代谢紊乱和慢性肾病）的发病机制有关的骨源性激素。假设：本提案的目的是检验分泌型蛋白激酶Fam 20 C通过磷酸化调节FGF 23的假设。该假设是基于三个最近的观察结果来制定的：1）Fam 20 C是以S-x-E/pS磷酸化分泌蛋白的蛋白激酶（Tagliabracci et.例如，Science 2012）。2)Fam 20 C缺失小鼠由于FGF 23的增加而发展低磷酸盐血症性佝偻病（Wang et.例如，PLoS基因组2012）和3）Fam 20 C在重要调控基序内的Ser 180和C末端内的3个高度保守氨基酸处磷酸化FGF 23，这可能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Fam 20 C调节FGF 23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组合来完成研究目标。指导阶段（K99，特定目标1和2）将包括体外和基于细胞的方法，以确定Fam 20 C调节FGF 23的分子机制。候选人将利用UCSD的资源和人员，这两者都是优秀的，非常适合该奖项的这一阶段。独立阶段（特异性目标3）将采用小鼠模型来研究Fam 20 C对FGF 23的调节。重要性：本申请中提出的实验将回答关于几种矿物质代谢紊乱的发病机制的基本问题，包括遗传性低磷酸盐血症性佝偻病;影响每20，000名新生儿中约1人。此外，这些结果将对慢性肾脏疾病患者产生重要影响，慢性肾脏疾病是一种日益严重的公共卫生流行病，影响着2600万美国人。培训内容：这项研究将在加州大学圣地亚哥分校（UCSD）药理学系进行，由Jack E.狄克逊。狄克逊博士是美国国家科学院院士、皇家学会会员，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白色博士和Joachim H.建立了合作。医学博士，著名肾病学家和流行病学家。候选人将通过UCSD进行课程，参加定期研讨会，并在国家科学会议上介绍他的研究结果。总的来说， 培训环境极佳，拟议的研究设计精良，富有创意。该假说解决了一个重要的问题，应该产生有影响力的结果，在内分泌学，肾脏学，心脏病学和基础生物医学研究。预计在获奖过程中发展的指导和研究技能将为候选人成功过渡到成为分泌蛋白磷酸化领域的杰出独立研究者和领导者提供坚实的基础。