Role of FAM20C in the Phosphorylation of SIBLING Protein

FAM20C 在 SIBLING 蛋白磷酸化中的作用

• 批准号: 8817274
• 负责人: XIAOFANG WANG
• 金额: $ 10.91万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8817274
• 关键词: Affect; Amino Acid Sequence; Biological Assay; C-terminal; Calvaria; Categories; Cell Culture System; Cells; Collagen Type I; Data; Defect; Dental Cementum; Dental Enamel; Dentin; Dentinogenesis; Disease; Enzymes; Event; Extracellular Matrix; Failure; Foundations; Future; Glycoproteins; Health; Hypophosphatemia; In Vitro; Incubated; Individual; Integrin Binding; Knowledge; Lead; Ligands; Link; Mass Spectrum Analysis; Minerals; Modality; Mus; Nodule; Odontoblasts; One-Step dentin bonding system; Osteoblasts; Osteogenesis; Peptide Hydrolases; Peptides; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotransferases; Physiologic calcification; Post-Translational Protein Processing; Protein Kinase; Proteins; Recombinants; Research Proposals; Rickets; Role; Serine; Site; Skeleton; Solid; Testing; Tissues; Tooth structure; Wild Type Mouse; Work; biomineralization; bone; bone sialoprotein; craniofacial complex; dentin matrix protein 1; mineralization; novel; osteopontin; protein aminoacid sequence; therapy development

DESCRIPTION (provided by applicant): This research proposal focuses on the role of FAM20C (also known as "dentin matrix protein 4" or "DMP4") in the phosphorylation of SIBLING proteins. Our recent data have established that FAM20C is essential to the formation and mineralization of bone and tooth, and that this protein kinase may be the primary enzyme responsible for catalyzing the phosphorylation of SIBLING proteins. We discovered: 1) FAM20C is highly expressed in the cells forming bone, dentin, cementum and enamel; 2) the inactivation of Fam20C in mice leads to rickets with extensive hypomineralization in murine bone; 3) Fam20C-KO mice have significant defects in the formation and mineralization of dentin, cementum and enamel; 4) the total phosphorylation level of non-collagenous proteins in the bone or dentin of Fam20C-KO mice is significantly lower than that of wild type mice. Furthermore, we and others revealed that recombinant FAM20C effectively phosphorylates some of the SIBLING proteins in vitro. Our preliminary data lead us to form the central hypothesis that FAM20C is the primary kinase catalyzing the phosphorylation of SIBLING proteins and the defects in the hard tissues of the Fam20C-KO mice may result from a significant loss of phosphorylation in the SIBLING proteins. To test this novel hypothesis, we propose the following three specific aims: (1) To determine the effects of FAM20C deficiency on the phosphorylation of SIBLING proteins. We will isolate OPN, BSP, the C-terminal fragment of DMP1, and DPP from the bone and dentin of Fam20C-KO mice and compare the phosphorylation level of these proteins with that in tissues from WT mice. We will also examine the effects of FAM20C on the phosphorylation of SIBLING proteins via 32P incorporation assay in the cultures of calvarial osteoblasts derived from Fam20C-KO and WT mice. (2) To examine phosphoserines in the SIBLING proteins isolated from the bone and dentin of Fam20C-KO and WT mice by digesting OPN, BSP, DMP1 C-terminal fragment, and DPP with proteases, separating the proteolytic peptides, and sequencing the isolated peptides with mass spectrometry. (3) To examine the effects of SIBLING proteins isolated from the Fam20C-KO mice on the mineralized nodule formation by osteoblasts and odontoblasts. The multipronged approaches proposed in this application will advance our understanding of the role of FAM20C in the phosphorylation of SIBLING proteins, and serve as the foundation for further elucidating the mechanism of biomineralization associated with the phosphorylation of SIBLINGs.

描述（由申请人提供）：该研究建议着重于FAM20C（也称为“牙本质基质蛋白4”或“ DMP4”）在同胞蛋白的磷酸化中的作用。我们最近的数据确定FAM20C对于骨和牙齿的形成和矿化至关重要，并且该蛋白激酶可能是负责催化同胞蛋白磷酸化的主要酶。我们发现：1）FAM20C在形成骨骼，牙本质，牙骨质和搪瓷的细胞中高度表达； 2）小鼠中FAM20C的失活导致鼠骨中具有广泛低矿化的rick鼠； 3）FAM20C-KO小鼠在牙本质，牙骨质和搪瓷的形成和矿化方面存在重大缺陷； 4）FAM20C-KO小鼠的骨骼或牙本质中非胶原蛋白的总磷酸化水平明显低于野生型小鼠的总磷酸化水平。此外，我们和其他人透露，重组FAM20C在体外有效地磷酸化了一些同胞蛋白。我们的初步数据导致我们形成了一个核心假设：FAM20C是催化同胞蛋白磷酸化的主要激酶，并且FAM20C-KO小鼠硬组织中的缺陷可能是由于同胞蛋白质中磷酸化的重大丧失而造成的。为了检验这一新的假设，我们提出以下三个特定目的：（1）确定FAM20C缺乏对同胞蛋白磷酸化的影响。我们将从FAM20C-KO小鼠的骨头和牙本中分离DMP1的OPN，BSP，DPP的C末端片段，并将这些蛋白的磷酸化水平与WT小鼠的组织中的磷酸化水平与该蛋白进行比较。我们还将检查FAM20C通过32P掺入分析在源自FAM20C-KO和WT小鼠的钙质成骨细胞培养物中对同胞蛋白磷酸化的影响。 （2）通过从FAM20C-KO和WT小鼠的骨骼和牙本质中分离出的同胞蛋白质中的磷酸，通过消化OPN，BSP，DMP1 C-末端片段，DPP和DPP用蛋白酶，分离蛋白水解肽，并与质谱分离蛋白酶，并与质谱分离。 （3）检查从FAM20C-KO小鼠中分离出的同胞蛋白对成骨细胞和odontoblasts矿化结节形成的影响。本应用程序中提出的多收益方法将提高我们对FAM20C在同胞蛋白质磷酸化中的作用的理解，并作为进一步阐明与兄弟姐妹磷酸化相关的生物矿化机制的基础。

项目成果

Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

具有序列相似性的家族成员 20C 是骨中小整合素结合配体 N 连接糖蛋白的主要但不是唯一的激酶。

• DOI: 10.1096/fj.15-273607
• 发表时间: 2016
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Yang,Xiudong;Yan,Wenjuan;Tian,Ye;Ma,Pan;Opperman,LynneA;Wang,Xiaofang
• 通讯作者: Wang,Xiaofang

Inactivation of Fam20B in Joint Cartilage Leads to Chondrosarcoma and Postnatal Ossification Defects.

• DOI: 10.1038/srep29814
• 发表时间: 2016-07-13
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Ma P;Yan W;Tian Y;Wang J;Feng JQ;Qin C;Cheng YS;Wang X
• 通讯作者: Wang X