Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation.
The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.
Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, 333 Cedar Street, New Haven, CT 06520-8024, USA.
Denis
D
Ostapenko
Janet L
JL
Burton
Ruiwen
R
Wang
Mark J
MJ
Solomon
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D018797:Cell Cycle Proteins; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054875:Ubiquitination
C516306:Acm1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D011494:Protein Kinases; C106494:HSL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae