Identification of Speg Binding Proteins and Phosphorylation Targets in Skeletal and Cardiac Muscle

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Title: Identification of Speg Binding Proteins and Phosphorylation Targets in Skeletal and Cardiac Muscle Abstract Severe life-threatening skeletal muscle and/or cardiac myopathies including centronuclear myopathy (CNM) and dilated cardiomyopathy are caused by mutations and/or deficiency of Speg (striated muscle preferentially expressed Ser/Thr protein kinase). Speg is at least partially located in triadic/dyadic junctional regions in skeletal and cardiac muscle. The mechanisms of Speg activation and Speg’s functional roles, binding proteins, and phosphorylation targets in skeletal and cardiac muscle remain to be fully elucidated. Current methods of assessing protein interacting partners suffer from lack of reproducibility and high levels of nonspecific interactions. This is particularly true for Speg. We created mice with a V5/HA tag inserted into Speg to use for anti-HA-Speg immunoprecipitations to identify Speg binding proteins. In this application, we will elucidate Speg interactome and identify Speg phosphorylation targets in the skeletal and cardiac muscle using optimized state-of-the-art mass-spec-associated proteomics techniques with parallel reaction monitoring (PRM). We will also identify phosphorylation sites on RyRs, Jph2 and Speg that are reduced by Speg deficiency. Our specific aims are to: SA 1: Define the effects of Speg deficiency on the muscle proteome. SA2: Identify Speg binding partners using mice with V5/HA tagged Speg. SA 3. Identify and quantify phosphorylation sites on RyRs, Jph2 and Speg and assess the effects of Speg deficiency on these sites. Successful completion of this study will identify Speg binding proteins and identify phosphorylation sites on its major targets. While this application centers on interactions and targets of Speg, the techniques developed will be applicable to other skeletal and cardiac muscle proteins.