Modulation of filamin C interactions and function by mechanical stress-induced phosphorylations

Abstract

When contracting, muscle cells are constantly exposed to mechanical stress with the resulting forces not only provoking unfolding and denaturation of proteins but also triggering cellular signaling processes, frequently leading to posttranslational modifications (PTMs). Among the most prominent PTMs numbers the phosphorylation of certain amino acids such as serine and threonine by protein kinases, counteracted by the versatile group of dephosphorylating phosphatases. Phosphorylations are known to trigger conformational changes in proteins, which in turn affect protein activity, e.g. by modulating binding sites for interaction partners. (De)phosphorylations of the actin-binding homodimeric protein filamin C (FLNC), predominantly expressed in skeletal muscle cells and the heart, were shown to modulate its properties and interactions. A recent quantitative phosphoproteomic analysis of cultivated muscle cells (C2C12) exposed to electrical pulse stimulation (EPS), to effectively model muscular exercise in vitro, revealed multiple stress-regulated phosphorylation sites in FLNC, implying potential regulatory effects of these modifications. Enhanced phosphorylation was clustered in the mechanosensory Ig-like domain 20, the dimerizing Ig-like domain 24 and was found throughout the FLNC molecule at serines flanking a highly conserved proline, which is part of the consensus PXSP motif recognized by MAP kinases. To analyze the effects of these PTMs, phosphorylation site mutants of FLNC were generated by replacing the relevant serines and threonines by the constitutively non-phosphorylatable alanine or the phosphomimetic aspartic acid. Proteolytic digestion assays of the latter in Ig-like domains 20 and 24 revealed affected protein stability pointing to altered conformations. In addition, phosphorylated FLNC variants of Ig-like domain 24 exhibited increased homodimer formation in chemical crosslinking assays, while the binding to the molecular chaperone HSPB7 was reduced, as shown biochemically by co-immunoprecipitations (CoIPs) and on the cellular level by bimolecular fluorescence complementation (BiFC) assays. EPS-treated C2C12 cells, transiently transfected with phosphomimetic mutants of FLNC, exhibited its reinforced translocation to myofibrillar lesions and a firmer integration into sarcomeric Z-discs during fluorescence recovery after photobleaching (FRAP) experiments. Taken together, these findings point to a phosphorylation-dependent “life cycle” of FLNC, relying on dynamic (de)phosphorylation events, which regulate the interaction of FLNC with the molecular chaperones HSPB1 and HSPB7, its homodimer formation and the stability of its integration into Z-discs. <br> The phosphorylation state of Ig-like domain 20 also had regulatory effects: next to the altered protein conformation, it also modulated interactions with molecular chaperones (HSPB7 and HSPB8) in CoIPs and resulted in a more stable association of FLNC to Z-discs in FRAP assays. The correspondingly reduced exchange rates of FLNC indicated a less efficient repair and accordingly a hampered compensation of mechanical cues, illustrated by its significantly increased translocation to myofibrillar lesions. Overall, this work highlights the necessity for dynamic phosphorylation and dephosphorylation cycles of FLNC during increased mechanical stress conditions to minimize deleterious consequences of contractions in cross-striated muscle cells.