Abstract 4340599: The Novel Muscle-specific RING Finger-2 Target Sorting Nexin 5 Regulates Protein kinase A Activity through Stabilization of Protein Kinase A Regulatory Subunit Type Iα and Controls Myogenic Differentiation

Abstract

Background: Muscle RING finger (MuRF) proteins are E3 ubiquitin ligases crucial for maintaining striated muscle homeostasis. MuRF1 is well known for its role in muscle atrophy, while MuRF2 and MuRF3 are involved in microtubule stabilization, influencing muscle differentiation and function. However, their cooperative functions remain unclear. This study aims to identify novel MuRF target proteins and investigate their roles in muscle cell regulation. Methods and Results: To identify interaction partners of MuRF2 and MuRF3, we performed stable isotope labeling with amino acids (SILAC) in cell culture followed by affinity purification and quantitative mass spectrometry (AP-MS). This approach allowed us to identify sorting nexin 5 (SNX5), a key component of the retromer complex responsible for retrograde vesicular transport, as a novel binding partner for both MuRF2 and MuRF3. Coimmunoprecipitation and immunocytochemistry confirmed the physical interaction between these proteins, with colocalization observed on early endosomes along microtubules. We further mapped the interaction domains and found that MuRF2 promoted SNX5 degradation through the ubiquitin-proteasome pathway, whereas MuRF3 counteracted this degradation. Mass spectrometry also identified the regulatory subunit of protein kinase A (RI-α) as cargo within SNX5-coated early endosomes in myocytes. To investigate the functional role of SNX5, CRISPR-Cas9 and siRNA experiments were performed. These studies showed that SNX5 stabilizes RI-α in muscle cells, regulating PKA activity. When SNX5 was deleted, PKA was activated, which led to the inhibition of the HDAC5-MEF2-Myostatin axis and impaired myogenic differentiation. Additionally, SNX5 influenced PKA activity and myogenic differentiation by promoting the degradation of HDAC5, which enhanced MEF2-dependent myostatin expression. Conclusion: MuRF2 and MuRF3 have opposing effects on SNX5-mediated retrograde transport, influencing PKA signaling and myogenic differentiation. SNX5 stabilizes RI-α within early endosomes, promoting proper myogenic differentiation. These findings expand the known functions of MuRF proteins beyond proteasomal degradation and identify SNX5 as a key regulator of PKA activity in muscle cells. These insights may lead to novel therapeutic targets for muscle-related disorders.