Poster Presentation 25th Annual Lorne Proteomics Symposium 2020

Crosstalk between protein methylation and phosphorylation in yeast and human (#104)

Joshua Hamey 1 , Daniela-Lee Smith 1 , Ryan J Separovich 1 , Marc R Wilkins 1
  1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia

Post-translational modifications (PTMs) are essential controllers of protein function within the cell. Phosphorylation and methylation, two of the most common PTMs, both regulate many cellular processes including cellular signaling, transcription, translation and chromatin remodeling. However, the extent of crosstalk between these PTMs remains largely unexplored. We have focused on two crucial aspects of PTM crosstalk: discovery of modifications pairs and characterisation of modifying enzyme specificity. First, through PTM enrichment, heavy methyl SILAC and mass spectrometric analysis in Saccharomyces cerevisiae, we uncovered new potential instances of crosstalk between methylation and phosphorylation. Phospho-methyl modification pairs mostly occurred on proteins associated with transcription and translation – these are highly connected proteins within the interactome. In particular, disordered glycine-arginine rich regions of proteins were subject to arginine methylation and serine phosphorylation. We showed that one such protein, yeast fibrillarin, is subject to crosstalk at serine-arginine-glycine-glycine (SRGG) motifs - methylation by the Hmt1 methyltransferase and phosphorylation by the Sky1 kinase were shown to be mutually exclusive. Phospho-methyl modification pairs were also discovered on the highly methylated elongation factor 1A (eEF1A) protein. This protein is targeted by five, highly specific methyltransferases, and novel phosphorylation events were discovered in close proximity to known lysine methylation sites. Moreover, deletion of eEF1A methyltransferases revealed the degree of crosstalk between these modifications. We also explored phospho-methyl crosstalk by investigating the mechanisms that underpin methyltransferase substrate recognition. To this end we developed and implemented methyltransferase motif analysis by mass spectrometry (MT-MAMS), a method to systematically characterise the amino acid sequences targeted by methyltransferases. Application of MT-MAMS to five yeast and human methyltransferases revealed known and likely crosstalk. In particular, we showed that Hmt1 is intolerant of acidic residues immediately upstream of the target arginine, reflective of its crosstalk with phosphorylation at these positions. As the roles of methylation and phosphorylation in the cell are further uncovered, it will be essential to determine the role that their crosstalk plays in determining their functions.