Functional analysis of PKLR p.Arg426Gln reveals isoform-specific aberrant PKR phosphorylation and PKL metabolic reprogramming

Pyruvate kinase deficiency (PKD) is an autosomal recessive hemolytic anemia caused by mutations in the PKLR gene, which encodes two tissue-specific isoforms: erythrocyte-specific PKR and hepatocyte-specific PKL. Although these isoforms share a common catalytic core and differ at their N-termini, how this N-terminal divergence shapes isoform-specific pathogenic mechanisms and contributes to the systemic manifestations of PKD remains unclear. Here, we investigated a PKD pedigree harboring a homozygous PKLR variant, NM_000298.6:c.1277G > A (p.Arg426Gln), to define its isoform-specific effects on PKR and PKL. Transcriptomic and proteomic profiling of the proband and healthy controls was used to identify mutation-associated pathway perturbations. Mechanistic studies employed isoform-specific cellular models (HEK293T for PKR-R426Q and THLE-2 for PKL-R395Q), site-directed mutagenesis, biochemical assays, and untargeted lipidomics. Zebrafish studies combined human PKLR mRNA expression, pharmacological rescue, and CRISPR/Cas9-mediated endogenous pklr disruption followed by rescue with human PKLR -WT or PKLR -R426Q mRNA. Functional analysis of the p.Arg426Gln mutation revealed distinct PKR- and PKL-associated pathogenic changes related to N-terminal divergence. Specifically, PKR-R426Q elicited PI3K-independent activation of PDPK1 and JAK2/STAT3 signaling, associated with increased AKT1 activation and aberrant phosphorylation involving the PKR-specific N-terminal S26 site. These alterations were accompanied by reduced PKR activity, glycolytic impairment, and MAPK suppression, and were partially ameliorated by pathway-directed pharmacological interventions in cellular models and human PKLR -R426Q mRNA-overexpressing zebrafish embryos. The glycolytic and erythroid phenotypes observed in h PKLR -R426Q mRNA-overexpressing zebrafish embryos were further supported by complementary genetic evidence from CRISPR/Cas9-mediated endogenous pklr disruption, in which the corresponding defects were rescued by human PKLR -WT but not PKLR -R426Q mRNA. Conversely, the homologous PKL-R395Q mutation exhibited phosphorylation-independent metabolic reprogramming, mitochondrial dysfunction, and lipid dysregulation. AG-348 improved PKR- and PKL-associated functional abnormalities in cellular and zebrafish models. Functional analysis suggests that N-terminal divergence contributes to the distinct pathogenic consequences of PKLR p.Arg426Gln in the PKR and PKL isoforms. The mutation appears to impair PKR mainly through aberrant phosphorylation-associated mechanisms, whereas PKL dysfunction is associated with phosphorylation-independent metabolic remodeling. These findings provide insight into genotype-phenotype correlations in PKD and highlight signaling and metabolic pathways that may warrant further therapeutic investigation.