Muscle RING-finger proteins (MuRF) regulate PKA activity via retrograde vesicular transport of RIα in skeletal muscle

N. Li (Greifswald)¹, E. Hammer (Greifswald)², Y. Li (Greifswald)³, U. Völker (Greifswald)², J. Fielitz (Greifswald)^4
1University Medicine Greifswald Department of Internal Medicine B, Cardiology, DZHK (German Center for Cardiovascular Research), partner site Greifswald Greifswald, Deutschland; ²Interfaculty Institute for Genetics and Functional Genomics Greifswald, Deutschland; ³University Medicine Greifswald, Clinic for Internal Medicine B The partner site Greifswald of the German Center of Cardiovascular Research (DZHK) Greifswald, Deutschland; ⁴Universitätsmedizin Greifswald Klinik und Poliklinik für Innere Medizin B Greifswald, Deutschland

Background:
Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins.
Methods and Results:
Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation.
Conclusion:
MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.