Targeting of a Novel Phosphorylation Site Prevents Disease Mutation‐associated Aggregation of Glial Fibrillary Acidic Protein (GFAP)
Authors: Kabiraj Parijat , Trogden Kathryn P , Snider Natasha T 
Published: November 2023

Tissue‐specific intermediate filament (IF) proteins, encoded by 73 human genes, form dynamic cytoskeletal and nucleoskeletal structures that confer cellular resistance to stress, regulate signaling, and organize the cell architecture. Missense mutations in IF genes that compromise filament assembly cause numerous human diseases, including neuropathies, myopathies, skin fragility disorders, metabolic dysfunctions, and premature aging syndromes. IF protein aggregation, a shared mechanism among IF‐associated diseases, is known to promote disease pathogenesis. Post‐translational modifications (PTMs) are attractive targets for restoring disease‐compromised IF proteostasis because PTMs regulate IF protein solubility, filament assembly, and protein interactions. The objective of this study was to identify and modulate key phosphorylation events involved in the aggregation of GFAP mutants that cause the rare leukodystrophy Alexander Disease (AxD), where GFAP is incorporated into toxic intracellular inclusions called Rosenthal fibers.MethodsWild‐type (WT) or AxD associated GFAP mutants (R79H, R79C, R88C, R239H, R239C, E373K, R416W) were expressed in SW13 vimentin‐negative human adrenal cancer cells, which lack endogenous cytoplasmic IFs. GFAP was analyzed by indirect immunofluorescence and confocal imaging, and biochemically by high salt extraction followed by isoelectric focusing (IEF) and mass spectrometry. Phosphorylation was modulated using site‐directed mutagenesis and pharmacologic kinase inhibition.ResultsAxD‐associated GFAP mutants formed prominent intracellular aggregates and promoted cell death. IEF analysis revealed charged GFAP species unique to the AxD mutants, but absent from the WT protein. MS proteomic analysis identified Serine 16 (S16), located in the N‐terminal “head” domain of GFAP, as a common modified residue among multiple AxD GFAP mutants. GFAP Ser‐16 is contained within a casein kinase (CK) motif that is present in the head domain of other IFs, pointing to a conserved regulatory mechanism. Site‐directed mutagenesis of S16 to a non‐phosphorylatable alanine (S16A) produced very short GFAP filaments, suggesting a critical role of this residue in GFAP filament assembly. Furthermore, a combined S16A/R79H mutation eliminated the profound GFAP aggregation of the R79H mutant and promoted GFAP protein solubility. Notably, the aggregation rescue mechanism of R79H following the S16A mutation involved hyperphosphorylation at other sites, as evidenced by the presence of at least 8 different charged species present in, and unique to, the double S16A/R79H mutant. A pharmacologic screen using the Tocriscreen™ kinase inhibitor toolbox identified several drugs that relieved GFAP R79H aggregation, most prominently inhibitors of MEK and EGFR, which are known to act upstream of CK.ConclusionAxD‐associated mutant GFAP aggregation can be blocked via selective targeting of a head domain serine phosphorylation site (Ser‐16). This phosphorylation event can be modulated at multiple levels to block GFAP aggregation and restore proper GFAP filaments.Support or Funding InformationSupported by NIH grants K01DK093776 and DK102450