Post-translational modifications (PTMs) can modulate protein-protein interactions. When used in combination, to modulate the specificity of interactions, PTMs can form “interaction codes”. To date, interaction codes have been characterised on a small number of proteins; histones, RNA polymerase II, FOXO transcription factors and chaperones. However, PTMs are known to be very widespread on eukaryotic proteins, raising the possibility that they may be a key means by which protein-protein interactions are regulated. We have termed this the “protein interaction code hypothesis”.

Finding new protein interaction codes is challenging, due to their complexity. Here we have simplified the study of interaction codes, by analysing paired PTMs, and through this defined a new interaction code based on an identical, recurring amino acid motif in yeast. The “SRGG” motif is found on 32 yeast proteins, many of which have functions in RNA maturation, transport or transcription. Phosphorylation and methylation, of “SRGG” motifs on yeast protein Npl3p has been shown to modulate protein-protein interactions. Kinase Sky1p and methyltransferase Hmt1p are the modifying enzymes. Here we describe a second yeast protein, Nop1p, to also be phosphorylated by kinase Sky1p and methyltransferase Hmt1p on “SRGG” motifs. Via the use of our conditional two-hybrid system we also show these modifications to modulate the protein-protein interactions of Nop1p.

The proximity of phosphorylation and methylation on “SRGG” motifs suggests these modifications may be subject to PTM crosstalk. Using in vitro peptide assays, coupled to LC-ETD MS/MS, we found that phosphorylation of “SRGG” attenuates the methylation by Hmt1p. However, prior methylation has little effect on Sky1p phosphorylation. Together these results characterise an “SRGG” protein interaction code, which recurs across yeast proteins Nop1p and Npl3p. We discuss how this code is likely to be important for nucleo-cytoplasmic shuttling.