24Otoferlin is essential for the fast and indefatigable release of synaptic vesicles at auditory inner hair 25 cell (IHC) ribbon synapses, being involved in exocytic, endocytic and regenerative steps of the synaptic 26 vesicle cycle. Serving diverse functions at this highly dynamic synapse implies that this multi-C2 domain 27 protein is precisely regulated. Here we found protein kinase C  (PKC) and otoferlin to colocalize in 28 endocytic recycling compartments upon IHC depolarization and to interact in an activity-dependent 29 manner. In vitro assays confirmed that PKC can phosphorylate otoferlin at five serine residues, which 30 correlates with increased serine phosphorylation in <40 nm proximity to otoferlin in murine IHCs that 31 can be fully blocked by combining PKC and CaMKII inhibitors. Moreover, otoferlin interacts with 32 calbindin-D28k in stimulated IHCs, which was precluded when PKC was inhibited. Similarly, the 33 activity-dependent increase in otoferlin-myosin VI interaction depends on PKC activation. We 34 propose that upon strong hair cell depolarization, PKC phosphorylates otoferlin, thereby enabling it 35 to interact with calbindin-D28k and myosin VI, building a Ca 2+ -dependent signaling complex that 36 possibly regulates different modes of endocytosis. 37 38 Keywords 39 calcium / inner ear / phosphorylation / ribbon synapse / synaptic transmission / 40 41 domain protein otoferlin seems to replace some of these proteins and is currently hypothesized to act 55 as the Ca 2+ sensor for exocytosis in mature IHCs (Roux et al, 2006; Michalski et al, 2017). Different 56 OTOF mutations lead to almost entirely abolished IHC exocytosis and thus to profound deafness in 57 humans and animal models (Yasunaga et al, 1999(Yasunaga et al, , 2000 Roux et al, 2006;Longo-Guess et al, 2007; 58 Marlin et al, 2010; Pangršič et al, 2010; Reisinger et al, 2011). Otoferlin is involved in vesicle priming 59 and fusion, vesicle replenishment, vesicle reformation from bulk endosomes, active zone clearance, 60 and clathrin-mediated endocytosis (Pangršič et al, 2010;Duncker et al, 2013;Jung et al, 2015; Strenzke 61 et al, 2016). It has been reported to interact with several proteins involved in the SV cycle, e.g. Rab8b, 62 myosin VI, CaV1.3 calcium channels, the adaptor protein 2 (AP-2) and endophilin A (Roux et al