APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex.
In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.
Tang
Z
Z
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, USA.
Li
B
B
Bharadwaj
R
R
Zhu
H
H
Ozkan
E
E
Hakala
K
K
Deisenhofer
J
J
Yu
H
H
D000595:Amino Acid Sequence; D000705:Anaphase; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; D002384:Catalysis; D002453:Cell Cycle; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004789:Enzyme Activation; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D015032:Zinc / Q000378:metabolism
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D021122:Protein Subunits; D025801:Ubiquitin; C105156:UBE2C protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C063109:ubiquitin-conjugating enzyme UBC4; C577351:ANAPC11 protein, human; D064173:Anaphase-Promoting Complex-Cyclosome; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; D008025:Ligases; D015032:Zinc