Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) β-Dependent Phosphorylation of GABA Triggers Lysosomal Degradation of GABA Receptors via Mind Bomb-2 (MIB2)-Mediated Lys-63-Linked Ubiquitination.

The G protein-coupled GABA receptors, constituted from GABA and GABA subunits, are important regulators of neuronal excitability by mediating long-lasting inhibition. One factor that determines receptor availability and thereby the strength of inhibition is regulated protein degradation. GABA receptors are constitutively internalized from the plasma membrane and are either recycled to the cell surface or degraded in lysosomes. Lys-63-linked ubiquitination mediated by the E3 ligase Mind bomb-2 (MIB2) is the signal that sorts GABA receptors to lysosomes. However, it is unknown how Lys-63-linked ubiquitination and thereby lysosomal degradation of the receptors is regulated. Here, we show that Ca/calmodulin-dependent protein kinase II (CaMKII) promotes MIB2-mediated Lys-63-linked ubiquitination of GABA receptors. We found that inhibition of CaMKII in cultured rat cortical neurons increased cell surface GABA receptors, whereas overexpression of CaMKIIβ, but not CaMKIIα, decreased receptor levels. This effect was conveyed by Lys-63-linked ubiquitination of GABA at multiple sites mediated by the E3 ligase MIB2. Inactivation of the CaMKII phosphorylation site on GABA(Ser-867) strongly reduced Lys-63-linked ubiquitination of GABA receptors and increased their cell surface expression, whereas the phosphomimetic mutant GABA(S867D) exhibited strongly increased Lys-63-linked ubiquitination and reduced cell surface expression. Finally, triggering lysosomal degradation of GABA receptors by sustained activation of glutamate receptors, a condition occurring in brain ischemia, was accompanied with a massive increase of GABA(Ser-867) phosphorylation-dependent Lys-63-linked ubiquitination of GABA receptors. These findings indicate that CaMKIIβ-dependent Lys-63-linked ubiquitination of GABA at multiple sites controls sorting of GABA receptors to lysosomes for degradation under physiological and pathological condition.