IKK regulates the deubiquitinase CYLD at the postsynaptic density

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• K63-linked polyubiquitination is crucial for protein trafficking in cellular pathways like endocytosis and autophagy.
• CYLD is a deubiquitinase targeting K63-linked polyubiquitins and is abundant at the postsynaptic density (PSD).
• Ca(2+)-dependent CaMKII activation of CYLD under excitatory conditions is known, but basal regulation was unclear.

Purpose of the Study:

• To investigate the regulation of CYLD at the PSD under basal conditions, independent of Ca(2+) signaling.
• To identify kinases involved in CYLD phosphorylation in isolated PSDs.
• To understand the role of IKK in basal CYLD activity and synaptic deubiquitination.

Main Methods:

• Utilized IKK and CaMKII inhibitors (IKK16, tatNEMO, CN21) to study CYLD phosphorylation in isolated PSDs and hippocampal cultures.
• Employed immuno-electron microscopy to visualize phosphorylated CYLD (pS-418) at the PSD.
• Performed in vitro phosphorylation assays with purified IKK and CYLD proteins.

Main Results:

• IKK, but not CaMKII, phosphorylates CYLD at the PSD under basal conditions, as shown by inhibitor studies.
• Phosphorylated CYLD (pS-418) is localized at the PSD under basal conditions, and its phosphorylation is inhibited by IKK16.
• IKK directly phosphorylates and activates CYLD, increasing K63-specific deubiquitinase activity in isolated PSDs.

Conclusions:

• Constitutive IKK activity at the PSD regulates CYLD in the absence of excitatory stimuli.
• This IKK-mediated regulation maintains basal levels of K63-linkage specific deubiquitination at the synapse.
• CYLD activity at the PSD is modulated by distinct kinase pathways under basal and excitatory conditions.