Dataset Information

Activity-Dependent Phosphorylation by CaMKII? Alters the Ca²⁺ Affinity of the Multi-C₂-Domain Protein Otoferlin.

ABSTRACT: Otoferlin is essential for fast Ca²⁺-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca²⁺/calmodulin-dependent serine/threonine kinase II delta (CaMKII?) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKII? in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKII? also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKII? revealed ten phosphorylation sites, five of which are located within C₂-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca²⁺ affinity of the recombinant C₂F domain 10-fold, and increases the Ca²⁺ affinity of the C₂C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKII? in IHCs.

SUBMITTER: Meese S

PROVIDER: S-EPMC5632675 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.

Meese Sandra S Cepeda Andreia P AP Gahlen Felix F Adams Christopher M CM Ficner Ralf R Ricci Anthony J AJ Heller Stefan S Reisinger Ellen E Herget Meike M

Frontiers in Synaptic Neuroscience 20171004

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otofer ...[more]

PMID: 29046633

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Project description:Residue Thr1604 in the CaV1.2 channel is a Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation site, and its phosphorylation status maintains the basic activity of the channel. However, the role of CaV1.2 phosphorylation at Thr1604 in myocardial hypertrophy is incompletely understood. Isoproterenol (ISO) was used to induce cardiomyocyte hypertrophy, and autocamtide-2-related inhibitory peptide (AIP) was added as a treatment. Rats in a myocardial hypertrophy development model were subcutaneously injected with ISO for two or three weeks. The heart and left ventricle weights, each of which were normalized to the body weight and cross-sectional area of the myocardial cells, were used to describe the degree of hypertrophy. Protein expression levels were detected by western blotting. CaMKII-induced CaV1.2 (Thr1604) phosphorylation (p-CaV1.2) was assayed by coimmunoprecipitation. The results showed that CaMKII, HDAC, MEF2 C, and atrial natriuretic peptide (ANP) expression was increased in the ISO group and downregulated by AIP treatment in vitro. There was no difference in the expression of these proteins between the ISO 2-week group and the ISO 3-week group in vivo. CaV1.2 channel expression did not change, but p-CaV1.2 expression was increased after ISO stimulation and decreased by AIP. In the rat model, p-CaV1.2 levels and CaMKII activity were much higher in the ISO 3-week group than in the ISO 2-week group. CaMKII-induced CaV1.2 channel phosphorylation at residue Thr1604 may be one of the key features of myocardial hypertrophy and disease development.Abbreviations: CaMKII: Ca2+/calmodulin dependent protein kinase II; p-CaMKII: autophosphorylated Ca2+/calmodulin dependent protein kinase II; CaM: calmodulin; AIP: autocamtide-2-related inhibitory peptide; ECC: excitation-contraction coupling; ISO: isoproterenol; BW: body weight; HW: heart weight; LVW: left ventricle weight; HDAC: histone deacetylase; p-HDAC: phosphorylated histone deacetylase; MEF2C: myocyte-specific enhancer factor 2C; ANP: atrial natriuretic peptide; PKC: protein kinase C.

Dataset Information

Activity-Dependent Phosphorylation by CaMKII? Alters the Ca²⁺ Affinity of the Multi-C₂-Domain Protein Otoferlin.

Publications

Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca<sup>2+</sup> Affinity of the Multi-C<sub>2</sub>-Domain Protein Otoferlin.

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Dataset Information

Activity-Dependent Phosphorylation by CaMKII? Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.

Publications

Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca<sup>2+</sup> Affinity of the Multi-C<sub>2</sub>-Domain Protein Otoferlin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Activity-Dependent Phosphorylation by CaMKII? Alters the Ca²⁺ Affinity of the Multi-C₂-Domain Protein Otoferlin.