Role of histone-lysine N-methyltransferase 2D (KMT2D) in MEK-ERK signaling-mediated epigenetic regulation: a phosphoproteomics perspective

Introduction Histone-lysine N-methyltransferase 2D (KMT2D) is an H3K4 methyltransferase and a potential tumor suppressor with a crucial role in regulating gene expression. Its dysregulation has been implicated in developmental disorders and several types of cancers. Despite this, the molecular mechanisms that govern its activity remain largely elusive. Among these, post-translational modifications, especially phosphorylation, serve as an essential regulator, fine-tuning KMT2D stability, localization and functional interactions for maintaining cellular homeostasis. With over 173 phosphorylation sites reported, KMT2D is significantly regulated by kinases and exploring its phospho-regulatory network based on targeted in vitro approaches is challenging. Methods We systematically curated and integrated the global phosphoproteomic datasets, along with their corresponding experimental conditions, to comprehensively identify the phosphorylation events reported for KMT2D. The site exhibiting the highest frequency of detection across these datasets is considered the predominant phosphorylation site. To investigate its functional significance, we analyzed the proteins and their phosphorylation sites that are differentially co-regulated with the predominant site, as well as its associated upstream kinases and interacting proteins. Results Among the 173 reported phosphorylation sites representing KMT2D, Serine 2274 (S2274) emerged as the predominant site being detected in over 42% of diverse mass spectrometry-based phosphoproteomics datasets. This site lies within one of KMT2D’s unique “ LSPPP ” motifs, suggesting a potential regulatory role. Detailed investigation on the differentially co-regulated protein phosphosites revealed the phosphorylation of KMT2D at S2274 is consistently and positively co-regulated with MAPK1/ERK2 activation, as well as with the proteins involved in the MAPK cascade, epigenetic regulation and cell differentiation. Notably, ERK2 was predicted as an upstream kinase targeting S2274, suggesting that KMT2D S2274 functions as a potential downstream effector of MEK-ERK signaling pathway, potentially linking to epigenetic regulation and cell differentiation. Further, our results highlighted a potential mechanistic link between disrupted phosphorylation at S2274 and the pathogenesis of Kabuki syndrome. Discussion This study delineates the phosphoregulatory network of KMT2D, positioning it as a dynamic epigenetic effector modulated by MEK-ERK signaling, with broader implications for cancer and developmental disorders.