Abstract
It has been reported that the Numb protein is methylated at lysine 158 and 163 and that this methylation is introduced by the SET8 protein lysine methyltransferase [Dhami et al., (2013) Molecular Cell 50, 565–576]. We studied this methylation in vitro using peptide arrays and recombinant Numb protein as substrates. Numb peptides and protein were incubated with recombinant SET8 purified after expression in E. coli or human HEK293 cells. However, no methylation of Numb by SET8 was detectable. SET8 methylation of Histone H4 and p53 peptides and proteins, which were used as positive controls, was readily observed. While SET8 methylation of Numb in cells cannot be ruled out, based on our findings, more evidence is needed to support this claim. It appears likely that another not yet identified PKMT is responsible for the reported methylation of Numb in cells.
Highlights
Histone post-translational modifications including acetylation, methylation and phosphorylation of several residues mainly in the histone tails are essential in modulating chromatin biology, gene expression and cellular development and they have important roles in diseases[1,2,3]
The sequence alignment of the lysine residues methylated in Numb (K158 and K163) with the SET8 reported substrates p53-K382 and H4K20 shows only limited sequence similarity and the reported Numb methylation sites differ at least at 4 out of 6 position from the SET8 preference (Fig. 1B)
Weak cysteine methylation by SET8 is not surprising since cysteine is the strongest nucleophile among all amino acid residues and cysteine methylation has been reported before for other AdoMet dependent methyltransferases, for example MLL119 or DNMT3A20. In summary these results show that recombinant SET8 expressed in E. coli cannot methylate the Numb peptides on peptide arrays while positive control peptides were methylated as expected
Summary
Histone post-translational modifications including acetylation, methylation and phosphorylation of several residues mainly in the histone tails are essential in modulating chromatin biology, gene expression and cellular development and they have important roles in diseases[1,2,3]. Similar to SET7/9, another well-characterized monomethyltransferase[14,15], the active pocket of SET8 is surrounded by tyrosine residues including Y334, which forms hydrogen bonds with the ε − amino group of lysine and prevents higher degrees of methylation. When this residue was exchanged to phenylalanine the mutated SET8 could introduce dimethylation at H4K2011. Since SET8 is a very specific PKMT with a long recognition sequence and it was found previously to function as a monomethyltransferase, we were interested to confirm that purified SET8 can dimethylate Numb peptides and protein in vitro
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