The unique N terminus of the UbcH10 E2 enzyme controls the threshold for APC activation and enhances checkpoint regulation of the APC.
In vitro, the anaphase-promoting complex (APC) E3 ligase functions with E2 ubiquitin-conjugating enzymes of the E2-C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2-C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2-E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N terminus increases substrate ubiquitination and the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N terminus to UbcH5 restricts ubiquitination activity but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2-E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.
Summers
Matthew K
MK
Department of Cellular Regulation, Genentech Inc., South San Francisco, CA 94080, USA.
Pan
Borlan
B
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080, USA.
Mukhyala
Kiran
K
Department of Bioinformatics, Genentech Inc., South San Francisco, CA 94080, USA.
Jackson
Peter K
PK
Department of Cellular Regulation, Genentech Inc., South San Francisco, CA 94080, USA; Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 94308, USA. Electronic address: pjackson@gene.com.
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
D004791:Enzyme Inhibitors; C105156:UBE2C protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D008239:Lysine