title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.
abstract: K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca(2+)-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca(2+) in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca(2+), but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Soe
authors.initials: S
authors.lastname: Thein
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Pham
authors.affiliation: Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.
authors.forename: K Ulrich
authors.initials: KU
authors.lastname: Bayer
authors.affiliation: EM Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Jung-Hwa
authors.initials: JH
authors.lastname: Tao-Cheng
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: dosemeca@mail.nih.gov.
authors.forename: Ayse
authors.initials: A
authors.lastname: Dosemeci
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D051550:I-kappa B Kinase / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D057907:Post-Synaptic Density / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D044767:Ubiquitin-Protein Ligases; D051550:I-kappa B Kinase; C581019:CYLD protein, rat; D043222:Ubiquitin Thiolesterase; D002118:Calcium
keywords: CYLD; Deubiquitinase; IKK; PSD; Postsynaptic density; Ubiquitin