INTEGRATED CHARACTERIZATION OF DUAL SPECIFICITY PHOSPHATASE 12 (DUSP12): FROM PUTATIVE REGULATION OF EIF6 TO MEDIATION OF CELLULAR ADAPTATION TO STRESS

Precise regulation of protein synthesis and ribosome biogenesis is fundamental for cellular homeostasis, growth, and adaptation. These energy-intensive processes are tightly controlled by signaling networks in which reversible phosphorylation plays a central role. A key node is eukaryotic initiation factor 6 (eIF6), a conserved anti-association factor that binds the 60S ribosomal subunit to prevent premature joining with the 40S subunit. Its release, required for 80S ribosome formation, is regulated by post-translational modifications, with phosphorylation of Ser235 emerging as a critical switch. Using non-phosphorylatable (S235A) and phosphomimetic (S235E) mutants, we showed that Ser235 phosphorylation is required for efficient eIF6 release and optimal translational output in vivo. However, unbiased biochemical approaches failed to identify stable upstream regulators of this modification, suggesting that eIF6 phosphorylation is controlled by transient or context-dependent interactions. Guided by proximity-labeling datasets, we focused on the atypical dual-specificity phosphatase DUSP12, a high-confidence proximity interactor of eIF6 previously implicated in ribosome biogenesis. Functional analyses revealed that DUSP12 overexpression enhances global protein synthesis, as measured by puromycin incorporation, without inducing major alterations in polysome profiles. Quantitative phosphoproteomics identified a broad set of DUSP12-dependent phosphorylation changes affecting nuclear proteins, ribosome biogenesis factors, and components of protein quality control, including the ER chaperone Calnexin and the nucleolar protein NOLC1. Comparative analyses further demonstrated that the C-terminal Zinc-Binding Domain (ZBD) of DUSP12 plays a critical role in shaping the downstream phosphoproteomic and proteomic landscape, Beyond its impact on translation-related pathways, DUSP12 overexpression was associated with a ZBD-dependent remodeling of the mitochondrial proteome, characterized by increased abundance of proteins involved in oxidative metabolism. This proteomic signature occurred in the absence of detectable changes in mitochondrial mass and was accompanied by functional metabolic alterations, including increased ATP levels and reduced lactate production. Integration with transcriptomic data revealed that these mitochondrial changes are largely uncoupled from corresponding transcriptional regulation, indicating a dominant contribution of post-transcriptional mechanisms. RNA sequencing instead revealed a limited and selective transcriptional response centered on stress-adaptive and signaling-related genes. Overall, this work indicates that DUSP12 influences cellular physiology through multilayered regulatory mechanisms operating predominantly downstream of transcription. Rather than functioning as a global transcriptional regulator, DUSP12 is positioned at the intersection of translational output, proteostasis, and mitochondrial metabolism in a domain-dependent manner, thereby contributing to cellular adaptation to biosynthetic and metabolic demands. These findings place DUSP12 as a regulatory node linking ribosome-associated processes to metabolic and stress-response pathways, with broader implications for understanding cellular adaptation in both physiological and pathological contexts.