Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca²⁺ dynamics linked to DEE66

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca²⁺ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca²⁺ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1–PACS2–PKD2 complex, regulating PKD2-dependent mitochondrial Ca²⁺ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca²⁺ uptake and the dramatic increase of the cytosolic Ca²⁺ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca²⁺-dependent neurotransmitter release.