N-terminal acetylation and methylation differentially affect the function of MYL9

Deciphering the histone code has illustrated that acetylation or methylation on the same residue can have analogous or opposing roles. However, little is known about the interplay between these post-translational modifications (PTMs) on the same nonhistone residues. We have recently discovered that N-terminal acetyltransferases (NATs) and N-terminal methyltransferases (NRMTs) can have overlapping substrates and identified myosin regulatory light chain 9 (MYL9) as the first confirmed protein to occur in either alpha-amino-methylated (N alpha-methyl) or alpha-amino-acetylated (N alpha-acetyl) states in vivo. Here we aim to determine if these PTMs function similarly or create different MYL9 proteoforms with distinct roles. We use enzymatic assays to directly verify MYL9 is a substrate of both NRMT1 and NatA and generate mutants of MYL9 that are exclusive for N alpha-acetylation or N alpha-methylation. We then employ eukaryotic cell models to probe the regulatory functions of these N alpha-PTMs on MYL9. Our results show that, contrary to prevailing dogma, neither of these modifications regulate the stability of MYL9. Rather, exclusive N alpha-acetylation promotes cytoplasmic roles of MYL9, while exclusive N alpha-methylation promotes the nuclear role of MYL9 as a transcription factor. The increased cytoplasmic activity of N alpha-acetylated MYL9 corresponds with increased phosphorylation at serine 19, a key MYL9 activating PTM. Increased nuclear activity of N alpha-methylated MYL9 corresponds with increased DNA binding. N alpha-methylation also results in a decrease of interactions between the N-terminus of MYL9 and a host of cytoskeletal proteins. These results confirm that N alpha-acetylation and N alpha-methylation differentially affect MYL9 function by creating distinct proteoforms with different internal PTM patterns and binding properties.