Involvement of two distinct ubiquitin E3 ligase systems for p27 degradation in corneal endothelial cells.
OBJECTIVE
p27(Kip1) (p27) is an important regulator of G(1) progression. For cells to proliferate, p27 must undergo proteolysis. FGF-2 enables phosphorylation of p27 at both the Thr-187 and Ser-10 sites, an event that is prerequisite for polyubiquitination. This study was undertaken to determine whether degradation of the two phosphorylated p27s is mediated by a distinct ubiquitin E3 ligase complex at different subcellular locations.


METHODS
Expression of p27, KPC1, KPC2, Skp1, Skp2, and Cul1 was analyzed by immunoblot analysis. Association of p27 with ubiquitin E3 ligase was determined with coimmunoprecipitation followed by immunoblot analysis. Inhibitors were used to inhibit proteasomal degradation and nuclear export of the phosphorylated p27. DNA synthesis was measured by BrdU incorporation into DNA.


RESULTS
Among ubiquitin ligase complex proteins, Cul1, KPC1, and KPC2 were constitutively expressed, whereas expression of Skp1 and Skp2 was temporally induced by FGF-2. Skp1, Skp2, and Cul1 were involved in polyubiquitination of phosphorylated p27 at Thr-187 (pp27Thr187) in nuclei. Maximum association of pp27Thr187 with the ubiquitin E3 ligase occurred 24 hours after FGF-2 stimulation. pp27Ser10 used the cytoplasmic ubiquitin E3 ligases KPC1 and KPC2, with maximum protein interaction observed at 8 hours. MG132 effectively blocked degradation of both pp27Thr187 and pp27Ser10, whereas leptomycin B blocked the nuclear export of pp27Ser10. Both inhibitors blocked BrdU incorporation into DNA.


CONCLUSIONS
The findings demonstrate distinct polyubiquitination pathways for pp27Thr187 and pp27Ser10; the former is ubiquitinated through the nuclear ubiquitin E3 ligase system during late G(1) phase; the latter by cytosolic ubiquitin E3 ligase during early G(1) phase.
Lee
Jeong Goo
JG
Doheny Eye Institute, Keck School of Medicine, University of Southern California, 1450 San Pablo Street, Los Angeles, CA 90033, USA.
Kay
EunDuck P
EP
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D044842:Cullin Proteins / Q000502:physiology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D044786:S-Phase Kinase-Associated Proteins / Q000502:physiology; D044843:SKP Cullin F-Box Protein Ligases / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D054875:Ubiquitination / Q000502:physiology*
D044842:Cullin Proteins; D015853:Cysteine Proteinase Inhibitors; D005231:Fatty Acids, Unsaturated; D007976:Leupeptins; D044786:S-Phase Kinase-Associated Proteins; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C038753:leptomycin B