A luminal kinase regulates sarcoplasmic reticulum calcium cycling

管腔激酶调节肌浆网钙循环

• 批准号: 9258219
• 负责人: Adam J Pollak
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258219
• 关键词: Adult; Affect; Arrhythmia; Binding; Biochemistry; Bone Development; Buffers; Calcium; Calcium Binding; Calcium-Binding Proteins; Calsequestrin; Cardiac; Cardiac Myoblasts; Cardiac Myocytes; Cardiovascular system; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular biology; Clinical; Communities; Complement; Cytosol; DNA Sequence Alteration; Data; Defect; Development; Disease; Dyes; Familial hypophosphatemic bone disease; Family; Genetic; Golgi Apparatus; Harvest; Heart; Heart Diseases; Histidine; Human; Knock-out; Knockout Mice; Label; Laboratories; Lead; Malignant Neoplasms; Metabolism; Modeling; Monitor; Mus; Muscle; Muscle Cells; Mutation; Myocardium; Organelles; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiological Processes; Play; Process; Property; Protein Kinase; Proteins; Rattus; Regulation; Reporting; Role; Sarcoplasmic Reticulum; Signal Transduction; Site; Spectrophotometry; Sudden Death; Testing; Tooth structure; Trees; Ventricular; Work; base; biomineralization; bone; casein kinase; cell type; comparative; experimental study; genetic regulatory protein; heart function; in vivo; interdisciplinary approach; interest; member; new therapeutic target; novel; phosphoproteomics; prevent; protein protein interaction; secretory protein; uptake

Project Summary/Abstract Protein phosphorylation by kinases is a ubiquitous cellular process and many diseases can be attributed to alterations in kinase function. Our laboratory recently discovered a kinase, Fam20C, which uniquely resides within the lumen of the secretory pathway. Its phosphorylation of secreted proteins is important for biomineralization of Ca2+ for bone and teeth formation, and mutations to Fam20C cause deadly defects to bone development and hypo-phosphatemic rickets. Recently, we showed that Fam20C is involved in several other physiological processes, including cancer development, and its myriad diverse roles are only beginning to be elucidated and appreciated. In fact, Fam20C is responsible for >90% of the secreted phosphoproteome. In our preliminary data here, we identified two Fam20C substrates, histidine-rich calcium binding protein (HRC) and calsequestrin 2 (CSQ2). They both reside within the lumen of the sarcoplasmic reticulum (SR), a secretory pathway organelle within cardiac muscle cells, which stores Ca2+ for muscle excitation-contraction. Proper heart function relies on precise Ca2+ cycling through the SR, and alterations to this process can cause heart disease, which is the world deadliest disease. A genetic mutation to HRC that causes heart disease has been previously identified. We show here that the mutation blocks Fam20C phosphorylation at that site. Furthermore, CSQ2 is also phosphorylated by Fam20C. The identity of a luminal SR kinase has been highly speculated, but never accurately reported. We hypothesize that Fam20C phosphorylation will regulate Ca2+ cycling through the SR. We will use a combination of biochemistry, cell biology, mouse genetics, and mass spectrophotometry to demonstrate this. With the development of a Fam20C specific cardiac mouse knockout, we will determine Fam20C's physiologic role in SR Ca2+ cycling. We will also use mass spectrophotometry to define the Fam20C dependent SR phosphoproteome. We anticipate that our experiments will reveal novel factors that control normal and diseased heart function, and will likely lead to new therapeutic targeting strategies.

项目总结/摘要 激酶引起的蛋白质磷酸化是一种普遍存在的细胞过程，许多疾病可以通过激酶磷酸化来治疗。 这归因于激酶功能的改变。我们的实验室最近发现了一种激酶Fam 20 C， 唯一地存在于分泌途径的管腔内。它对分泌蛋白的磷酸化作用是 重要的生物矿化钙2+的骨和牙齿的形成，突变Fam 20 C的原因 致命的骨骼发育缺陷和低磷酸盐血症性佝偻病。最近，我们发现， Fam 20 C还参与其他几种生理过程，包括癌症的发展， 无数不同的角色才刚刚开始被阐明和理解。事实上，Fam 20 C是 负责>90%的分泌磷酸化蛋白质组。 在我们的初步数据中，我们鉴定了两种Fam 20 C底物，富组氨酸钙结合 蛋白（HRC）和钙螯合蛋白2（CSQ 2）。它们都存在于肌浆细胞的腔中 网状细胞（SR），心肌细胞内的分泌途径细胞器，为肌肉储存Ca 2 + 兴奋收缩正常的心脏功能依赖于通过SR的精确的Ca 2+循环， 这一过程的改变会导致心脏病，这是世界上最致命的疾病。遗传 导致心脏病的HRC突变先前已被确定。我们在这里展示的 突变阻断Fam 20 C在该位点的磷酸化。此外，CSQ 2也被磷酸化， Fam20C。管腔SR激酶的身份已被高度推测，但从未准确报道。 我们假设Fam 20 C磷酸化将通过SR调节Ca 2+循环。 使用生物化学、细胞生物学、小鼠遗传学和质谱法的组合， 演示一下。随着Fam 20 C特异性心脏敲除小鼠的发展，我们将 确定Fam 20 C在SR Ca 2+循环中的生理作用。我们还将使用质量分光光度法， 定义Fam 20 C依赖的SR磷酸化蛋白质组。我们预计我们的实验将揭示 控制正常和患病心脏功能的新因素，并可能导致新的治疗方法， 瞄准战略。