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Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

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.

SUBMITTER: Weirich S

PROVIDER: S-EPMC4585732 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

Weirich Sara S Kusevic Denis D Kudithipudi Srikanth S Jeltsch Albert A

Scientific reports 20150922

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 ...[more]

PMID: 26391684

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Project description:Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic ?-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

Dataset Information

Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

Publications

Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

Similar Datasets

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