Role of Histone-lysine N-methyltransferase 2D (KMT2D) in MEK-ERK signaling-mediated epigenetic regulation: A Phosphoproteomics perspective

Sreeshma Ravindran KammarambathLeona D’cunhaAthira Perunelly GopalakrishnanAmal FahmaNeelam KrishnaAlthaf MahinSamseera UmmarPrathik Basthikoppa ShivamurthyInamul Hasan Madar^*Rajesh Raju^*

• Yenepoya (Deemed to be University), Mangaluru, India

Histone-lysine N-methyltransferase 2D (KMT2D) is an H3K4 methyltransferase and a potential tumor suppressor with crucial role in regulating gene expression, its dysregulation has been implicated in developmental disorders and several types of cancer. 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's 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. Hence, we systematically assembled the global phosphoproteomics datasets along with their experimental conditions to capture the phosphorylation events reported for KMT2D. Among the phosphorylation sites identified in KMT2D, Serine 2274 (S2274) emerged as the most 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. To further explore its functional significance, we analyzed proteins and their phosphorylation site that are differentially co-regulated with S2274, along with its associated kinases and interacting partners. Detailed investigations revealed that the phosphorylation at KMT2D at S2274 is consistently and positively co-regulated with MAPK1/ERK2 activation, as well as with the proteins involved in 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. Overall, 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.