Ser(120) of Ubc2/Rad6 regulates ubiquitin-dependent N-end rule targeting by E3{alpha}/Ubr1.
In CHO cells, CDK1/2-dependent phosphorylation of Ubc2/Rad6 at Ser(120) stimulates its ubiquitin conjugating activity and can be replicated by a S120D point mutant (Sarcevic, B., Mawson, A., Baker, R. T., and Sutherland, R. L. (2002) EMBO J. 21, 2009-2018). In contrast, we find that ectopic expression of wild type Ubc2b but not Ubc2bS120D or Ubc2bS120A in T47D human breast cancer cells specifically stimulates N-end rule-dependent degradation but not the Ubc2-independent unfolded protein response pathway, indicating that the former is E2 limiting in vivo and likely down-regulated by Ser(120) phosphorylation, as modeled by the S120D point mutation. In vitro kinetic analysis shows the in vivo phenotype of Ubc2bS120D and Ubc2bS120A is not due to differences in activating enzyme-catalyzed E2 transthiolation. However, the Ser(120) mutants possess marked differences in their abilities to support in vitro conjugation by the N-end rule-specific E3α/Ubr1 ligase that presumably accounts for their in vivo effects. Initial rate kinetics of human E3α-catalyzed conjugation of the human α-lactalbumin N-end rule substrate shows Ubc2bS120D is 20-fold less active than wild type E2, resulting from an 8-fold increase in K(m) and a 2.5-fold decrease in V(max), the latter reflecting a decreased ability to support the initial step in target protein conjugation; Ubc2bS120A is 8-fold less active than wild type E2 due almost exclusively to a decrease in V(max), reflecting a defect in polyubiquitin chain elongation. These studies suggest a mechanism for the integrated regulation of diverse ubiquitin-dependent signaling pathways through E2 phosphorylation that yields differential effects on its cognate ligases.
Department of Biochemistry and Molecular Biology and the Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Brajesh
B
Kumar
Kimberly G
KG
Lecompte
Jennifer M
JM
Klein
Arthur L
AL
Haas
D000818:Animals; D002384:Catalysis; D002417:Cattle; D045744:Cell Line, Tumor; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
D012694:Serine; D007768:Lactalbumin; C471545:UBE2A protein, human; D044763:Ubiquitin-Conjugating Enzymes; C113474:UBR1 protein, human; D044767:Ubiquitin-Protein Ligases