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Q. Liu, F. Shang, S. G. Rhee, A. Taylor; Ubc3 Cooperates With APC in a Novel Way to Control the G2/M Transition. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4235.
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Ubc3/Cdc34 is an ubiquitin-conjugating enzyme (Ubc) with well established functions in the G1-to-S-phase transition. In our previous report, we found that over expression of mutant (mt) and wild type (wt) Ubc3 delayed the HLEC cell cycle in metaphase, rather than the expected G1- phase transition. Expression of both Ubc3s also stabilized M-phase regulators, cyclin A, cyclin B, and securin. This implied that Ubc3 was blocking the activity of the E3, called the APC, that regulates the G2/M transition. To test the novel hypothesis that Ubc3 cooperates with APC to control the G2/M transition, we determined if Ubc3 blocks the interaction of the APC with its usual catalytic partner Ubc10.
Bead-immobilized GST-APC11, the active subunit of the APC, was incubated with His6-Ubc10 with or without His6-Ubc3; the beads were then washed and subjected to immunoblot analysis with antibodies to Ubc3 and Ubc10 to observe displacement of Ubc10 by Ubc3. To explore the specific mechanism how Ubc3 might delay cell cycle in metaphase, lysate was prepared from nocodazole synchronized, mitosis phase, HeLa cells and the degradation of endogenous cyclin B1 was measured in a reaction mixture containing Ubc10, in the absence or in the presence of Ubc3 and in the ubiquitin and cell lysate. The reactions were performed and subjected to immunoblot analysis with antibodies to the cyclin B.
Ubc3 competes with Ubc10 for APC11 in a concentration-dependent manner.Ubc3 prevents destruction of cyclin B. However, cyclin B degradation was not affected by Ubc2.
We discovered a previously undescribed Ubc3-APC interaction which interferes with the usual binding of Ubc10 to the APC. This results in a block of the degradation of G2/M substrates, resulting in metaphase arrest.
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