GCN5-Catalyzed WSTF Benzoylation Activates Tumor Glycolysis
DOI:
https://doi.org/10.71204/pk2jdq31Keywords:
WSTF, GCN5, Benzoylation, Cancer, GlycolysisAbstract
Williams syndrome transcription factor (WSTF), traditionally recognized as a nuclear-localized transcription factor and histone tyrosine kinase, has been detected in the cytoplasm of various cancer cells in our previous observations. This finding suggests the existence of an uncharacterized nucleocytoplasmic shuttling mechanism and extranuclear functions of WSTF. This study aims to investigate whether WSTF’s subcellular localization is regulated by novel post-translational modifications, and to clarify its cytoplasmic functions and role in tumor metabolic reprogramming. Through post-translational modification proteomics analysis, we identified lysine benzoylation at position 181 (K181) of WSTF. After knocking down candidate enzyme genes individually with specific siRNAs, WB analysis revealed a significant decrease in WSTF-K181 benzoylation levels only when GCN5 was knocked down, suggesting that GCN5 is a potential enzyme catalyzing this modification. Functional assays demonstrated that K181 benzoylation is a key driver for WSTF translocation from the nucleus to the cytoplasm. Furthermore, 4D-Labelfree tyrosine phosphorylation proteomics unexpectedly identified hexokinase 1 (HK1) as a novel cytoplasmic substrate of WSTF. In vitro and in vivo experiments verified that WSTF directly binds to HK1 and phosphorylates it at tyrosine 667, thereby enhancing HK1 kinase activity and promoting glucose uptake and lactate production. Functionally, expression of wild-type WSTF significantly promoted the proliferation, migration, and in vivo tumorigenicity of breast cancer cells, while the kinase-inactive mutant (C338A) or benzoylation-deficient mutant (K181A) remarkably attenuated these oncogenic effects of WSTF. This study is the first to reveal a novel mechanism by which GCN5-mediated benzoylation regulates WSTF nucleocytoplasmic shuttling. Additionally, we discovered that WSTF drives tumor glycolysis in a non-canonical manner by directly phosphorylating the metabolic enzyme HK1. Our findings break through the traditional understanding of WSTF functions and provide a new perspective for deciphering tumor metabolic reprogramming.
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