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(Investigative Ophthalmology and Visual Science. 2008;49:189-196.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0855
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Involvement of Two Distinct Ubiquitin E3 Ligase Systems for p27 Degradation in Corneal Endothelial Cells

Jeong Goo Lee1 and EunDuck P. Kay1,2

1From the Doheny Eye Institute and the 2Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California.

PURPOSE. p27Kip1 (p27) is an important regulator of G1 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 G1 phase; the latter by cytosolic ubiquitin E3 ligase during early G1 phase.






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