Molecular Characterization of Propionyllysines in Non-histone Proteins

Zhongyi Cheng,Yi Tang,Yue Chen,Sungchan Kim,Huadong Liu,Shawn S.C Li,Wei Gu,Yingming Zhao

doi:10.1074/mcp.m800224-mcp200

Abstract

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones. The molecular components involved in the two protein modification pathways are unknown, hindering further functional studies. Here we report identification of the first three in vivo non-histone protein substrates of lysine propionylation in eukaryotic cells: p53, p300, and CREB-binding protein. We used mass spectrometry to map lysine propionylation sites within these three proteins. We also identified the first two in vivo eukaryotic lysine propionyltransferases, p300 and CREB-binding protein, and the first eukaryotic depropionylase, Sirt1. p300 was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation, like lysine acetylation, is a dynamic and regulatory post-translational modification. Based on these observations, it appears that some enzymes are common to the lysine propionylation and lysine acetylation regulatory pathways. Our studies therefore identified first several important players in lysine propionylation pathway.

Highlights

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones
Lysine propionylation is originally identified in histones [12], but whether it, like lysine acetylation, is present in non-histone proteins remains unknown, as do the identities of the enzymes responsible for regulating the modification
We show that [1] lysine propionylation is present in three nonhistone proteins (p53, p300, and CBP); [2] p300 and CBP can catalyze lysine propionylation of p53; [3] p300 and CBP have auto-lysine propionylation activities; and [4] Sirt1 is a depropionylase that removes lysine propionylation from p53 and p300

Summary

MATERIALS AND METHODS

Materials: Plasmids, Antibodies, and Other Reagents—Plasmids used in this study were described previously [12, 22]. p300 HAT-dead mutant, p300DY, was generated by mutation of aspartic acid in 1399 to tyrosine; CBP HAT-dead mutant, CBP-LD, was generated by mutation of both leucine in 1345 and aspartic acid in 1346 to alanine; Sirt, enzymatic defective mutant was generated by replacing histidine in 363 by tyrosine. Detection of Lysine Propionylation of p53, p300, and CBP in Cells—Twenty-four hours after transfection with various plasmids, as indicated, transfected cells were treated with described HDAC inhibitors for 6 h, harvested, and lysed in FLAG-lysis buffer (50 mM TrisHCl, pH 7.9, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 10% glycerol, and fresh protease inhibitor mixture (Sigma)) plus 10 mM sodium butyrate. Propionylation of p53 in the total cell extracts or in material immunoprecipitated with M2-agarose beads was detected by Western blotting analysis using the antipropionyllysine antibody described above. Propionylation of p300 or CBP in material immunoprecipitated with M2 or HA-agarose beads was detected by Western blot analysis using the anti-propionyllysine antibody. All identified peptides bearing modifications were manually inspected according to a procedure we described previously [21]

RESULTS

DISCUSSION