Investigating post-translational modifications and novel interaction partners of otoferlin

von Andreia Filipa Pereira Cepeda
Dissertation
Datum der mündl. Prüfung:2019-10-21
Erschienen:2019-10-30
Betreuer:PD Dr. Ellen Reisinger
Gutachter:PD Dr. Ellen Reisinger
Gutachter:Prof. Dr. Silvio O. Rizzoli
Gutachter:Prof. Dr. Manuela Schmidt
Gutachter:Prof. Dr. Henning Urlaub
Gutachter:PD Dr. Sven Thoms
Gutachter:Prof. Dr. Ralf Heinrich
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-7705

 

 

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Name:Andreia Cepeda - PhD Dissertation.pdf
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Otoferlin is a large multi-C2-domain protein essential for hearing and fast Ca2+-triggered transmitter release from auditory IHCs. Mutations in the OTOF gene are linked to a form of autosomal recessive non-syndromic hearing loss, DFNB9. Otoferlin is involved in several steps of the synaptic vesicle cycle in IHCs including vesicle fusion, vesicle reformation, vesicle recycling, endocytosis and coupling of exo- and endocytosis. While some progress has been made in understanding its role in IHC synaptic transmission, mechanisms regulating its function have not been studied to date. Second messenger-activated protein kinases regulate synaptic transmission in conventional synapses via phosphorylation of presynaptic proteins thereby controlling presynaptic plasticity, protein interactions within the release apparatus, endocytosis and trafficking events. In this thesis, I focused on deciphering the role of protein kinases in IHC synaptic transmission. Together with my collaborators, I showed for the first time that synaptic activity in IHC synapses is also regulated by phosphorylation of presynaptic proteins. By combining immunohistochemistry, in situ proximity ligation assays (PLAs), confocal microscopy, real-time PCR, mutagenesis, microscale thermophoresis (MST), pull-downs, co-immunoprecipitations (co-IPs), in vitro assays and mass spectrometry approaches, we showed that Ca2+/calmodulin-dependent serine/threonine kinase delta (CaMKIIδ) and protein kinase C α (PKCα) phosphorylate otoferlin and regulate its function in rodent IHCs. CaMKIIδ and PKCα are expressed throughout the cell and both revealed to be in close proximity to otoferlin upon strong stimulation. Physical association between the two kinases and otoferlin was confirmed via binding assays, and kinase-specific phosphorylation sites were retrieved: CaMKIIδ phosphorylates otoferlin in its C2 domains (C2C, C2D, C2de, C2F) whereas PKCα seems to target linker regions and the FerA domain (presumed to be involved in membrane-association events), suggesting a combined but distinct action of CaMKIIδ and PKCα. Phosphorylation by CaMKIIδ affects the affinity of otoferlin’s C2C and C2F domains to Ca2+ under physiological conditions. PKCα is targeted upon activation (either pharmacologically or following high K+ stimulation) to the basolateral plasma membrane and to endocytic compartments where it interacts with otoferlin. The previously reported interaction of otoferlin with myosin VI appears to be PKC-dependent. Moreover, otoferlin interacts with the EF-hand protein calbindin-D28k in a PKC-dependent manner, whereas PKCα and calbindin-D28k seem not to interact directly. The association of these three proteins probably happens in a sequential fashion and potentially regulates different modes of membrane internalization and may control the dynamics of the synaptic vesicle cycle in IHCs. The PKC-dependent association of otoferlin with calbindin-D28k is especially potentiated under strong stimulatory conditions and might play a role in clathrin-independent events like ultrafast endocytosis. This mechanism may constitute a molecular switch between different modes of endocytosis, thus providing the grounds for fast and efficient vesicle recycling, hallmarks of IHC ribbon synapses.
Keywords: otoferlin; CaMKII; PKC; ribbon synapse; phosphorylation
 
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