A luminal kinase regulates sarcoplasmic reticulum calcium cycling

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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.