A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination.
Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.
Podust
V N
VN
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Brownell
J E
JE
Gladysheva
T B
TB
Luo
R S
RS
Wang
C
C
Coggins
M B
MB
Pierce
J W
JW
Lightcap
E S
ES
Chau
V
V
D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D020134:Catalytic Domain; D018797:Cell Cycle Proteins; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003545:Cysteine; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000075687:NEDD8 Protein; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D025521:Tumor Suppressor Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D019927:Cyclin E; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012694:Serine; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D008025:Ligases; D003545:Cysteine