Epidermal growth factor (EGF) is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. Its receptor EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here, we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, we found PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, distinct from PKM2, we observed that the association of additional phosphotyrosine-binding proteins with OGT was also enhanced when Y976 was phosphorylated. These proteins included STAT1, STAT3, STAT5, PKCδ, and p85, which are reported to be O-GlcNAcylated. Together, we show EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and propose that this posttranslational modification might be important for substrate selection by OGT.

表皮生长因子（EGF）是特征最明确的生长因子之一，在细胞增殖和分化中起着至关重要的作用。其受体 EGFR 已被广泛探索为针对多种癌症的治疗靶点，例如肺癌和胶质母细胞瘤。最近的研究已经建立了失调的 EGF 信号传导和代谢重编程之间的联系，特别是有氧糖酵解中的重新布线，这也被称为 Warburg 效应，并被认为是癌症的标志。丙酮酸激酶 M2 (PKM2) 是一种限速酶，控制糖酵解的最后一步，是 Warburg 效应的主要调节剂。我们之前展示了 PKM2 T405/S406 O-GlcNAcylation，对 PKM2 去四聚化和活性很重要的关键标记，被 EGF 显着上调。然而，EGF 调节 PKM2 O -GlcNAcylation 的机制仍未确定。在这里，我们证明 EGF通过刺激 OGT Y976 磷酸化促进O -GlcNAc 转移酶 (OGT) 与 PKM2 结合。因此，我们发现 PKM2 O -GlcNAcylation 和去四聚化被上调，导致 PKM2 活性显着降低。此外，与 PKM2 不同，我们观察到当 Y976 被磷酸化时，额外的磷酸酪氨酸结合蛋白与 OGT 的关联也得到了增强。这些蛋白质包括 STAT1、STAT3、STAT5、PKCδ 和 p85，据报道它们是O-GlcNA酰化。总之，我们表明 EGF 依赖性 Y976 磷酸化对 OGT-PKM2 相互作用至关重要，并提出这种翻译后修饰可能对 OGT 选择底物很重要。