N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome.
Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.
Coulombe
Philippe
P
Institut de recherche en immunovirologie et cancérologie, Department of Molecular Biology, Université de Montréal, Quebec, Canada.
Rodier
Geneviève
G
Bonneil
Eric
E
Thibault
Pierre
P
Meloche
Sylvain
S
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016324:Sequence Tagged Sites; D025801:Ubiquitin / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D009097:Multienzyme Complexes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine