Functions and regulation of synaptopodin-2 isoforms under mechanical stress in muscle cells

Abstract

Synpo2 isoforms are adapter proteins in myofibrillar Z-disc of striated muscle engaged in the homeostasis of the actin-crosslinking protein filamin C under mechanical stress. They interact with the co-chaperone BAG3, which is involved in CASA-mediated degradation of unfolded and damaged protein. In this study isoform-specific functions of Synpo2b, containing a PDZ- domain and Synpo2e lacking this domain, were analyzed. Under mechanical stress Synpo2e was less stably associated with Z-discs than Synpo2b and in part transferred to myofibrillar lesions. Synpo2e was also shown to recruit BAG3 to Z-discs and lesions via interactions of its proline-rich motif with the WW domain of the co-chaperon. This study provides evidence for a role of myofibrillar lesions as a quality control compartment essential for repair of damaged myofibrils with Synpo2 as a key proteostasis factor crucial for the maintenance of the contractile apparatus.<br /> Based on phosphoproteome data pointing to a possible role of (de)phosphorylation in regulating Synpo2, phosphomimetic mutants were generated. A cluster of three phosphosites in the H2 region of Synpo2 revealed a regulatory function in its interactions with filamin C and a-actinin. The mutant mimicking the phosphorylated state of the triple phosphosite bound less strongly to both proteins. The less stable interaction with the actin cytoskeleton led to increased lesion formation, and considerable recruitment of Synpo2b to these structures. Hence, the triple phosphosite has a strong impact on the activity and localization of Synpo2b under mechanical stress. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated increased stability of constitutively dephosphorylated Synpo2b affecting the turnover rate of the protein, thus confirming an influence of this phosphosite on its dynamics. In FRAP experiments constitutively dephosphorylated Synpo2e showed a higher affinity of Synpo2e for Z-discs, whereas the turnover of the protein remained unchanged and was independent of the phosphorylation state. Taken together, it is evident that these clustered phosphorylation sites have strong regulatory roles for the localization, mobility, dynamics, and protein-protein interactions of both investigated Synpo2 isoforms.<br /> LC3B builds a scaffold to recruit numerous proteins on the surface of autophagosomes and interacts via its LIR specific peptide motif. Database analyses identified a LIR motif in the Synpo2 H1 region, which also contains a phosphosite in its core sequence. A peptide array using LIR phosphosite peptides showed a phosphorylation-dependent interaction of Synpo2 with LC3B. In contrast, bimolecular fluorescence complementation assays using LIR phosphosite mutants revealed no phosphorylation-dependent interaction of LC3B and Synpo2. Synpo2e, which is usually localized in Z-discs and lesions, is at least in part associated with vesicular structures in the constitutively phosphorylated state of the LIR motif. These results suggest that the localization and function of this isoform may be affected by LIR motif phosphorylation. FRAP experiments with Synpo2b variants showed a significantly reduced half-life of the dephosphorylation mimicking mutant compared to both the wildtype and the phosphorylation mimicking mutant. Hence it is evident that the LIR phosphosite has a regulatory role for the localization, mobility, dynamics, and the interactions with LC3B of both investigated Synpo2 isoforms.