Pseudokinase-converting mutation in protein kinase C alpha drives chordoid glioma by pathway rewiring.

Chordoid glioma (ChG) is a rare, low-grade brain tumor characterized by a novel recurrent point mutation, D463H, in the kinase domain of protein kinase C alpha (PKCα). The mutation is invariably an Asp to His substitution, suggesting a unique function beyond catalytic inactivation associated with other cancer-associated PKCα mutations. Here, we show that this mutation converts PKCα into a pseudokinase, abolishing catalytic activity, and, additionally, confers novel scaffolding functions. Activity assays in vitro and in cellulo revealed that PKCαD463H is catalytically inactive and functions as a dominant-negative to suppress endogenous PKC activity. Molecular dynamics simulations predicted that mutation to His, but not Asn, not only destabilizes the active site, but stabilizes the substrate-binding helices in the kinase C-lobe to potentially promote aberrant interactions. Supporting this, phosphoproteomic, proximity labeling, and coimmunoprecipitation mass spectrometry data from cells overexpressing PKCαD463H identified both altered phosphorylation of substrates and binding to multiple proteins involved in cell-cell junctions compared to WT enzyme. Last, single nuclei RNAseq established that ChG derives from specialized tanycytes. Our data reveal that this disease-defining, fully penetrant mutation converts PKCα into a pseudokinase with novel scaffold functions that uniquely rewire the cellular interactome to impair cell junction function.