Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

Abstract

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.

The checkpoint transcriptional response: Make sure to turn it off once you are satisfied

“…The APC coactivator Cdh1, which is involved in the recruitment of target substrates to the APC, is inhibited from binding its targets by Cdc28-dependent stabilization of Acm1. 69 TOS4, MCD1, SWE1 and ACM1 are cell cycle-regulated, and, corroborating our hypothesis that switch genes expression needs to be tightly regulated, overexpression of these genes was linked to growth defect and/or cell cycle arrest. 65,66 Further analysis of the other switch genes, such as the TOS genes and eight putative ORFs, will show their potential role as checkpoint effectors.…”

Acm1 contributes to nuclear positioning by inhibiting Cdh1-substrate interactions

“…16 Conditions were identical to those under which we observed the spindle defects in Figure 1. Acm1 has been shown to block the binding of Clb2 and Hsl1 to Cdh1, 7,9,10,22 and ubiquitination of several of these substrates by APC Cdh1 is inhibited by Acm1 in vitro. 7,9,22 However, our results here argue that Acm1 is not required for APC Cdh1 inhibition in vivo, even in anaphase, when Cdk activity has been lowered by cyclin degradation.…”

“…Acm1 has been shown to block the binding of Clb2 and Hsl1 to Cdh1, 7,9,10,22 and ubiquitination of several of these substrates by APC Cdh1 is inhibited by Acm1 in vitro. 7,9,22 However, our results here argue that Acm1 is not required for APC Cdh1 inhibition in vivo, even in anaphase, when Cdk activity has been lowered by cyclin degradation. 23 It was previously suggested that Acm1's pseudosubstrate properties could be important for preventing inappropriate Cdh1-substrate interactions.…”

“…[7][8][9][10] Acm1 inhibits the ubiquitin ligase activity of APC Cdh1 in vitro and overexpression of Acm1 restores viability to strains expressing toxic levels of Cdh1, suggesting that it can inhibit APC Cdh1 activity in vivo. However, no cell cycle defects have been observed for strains lacking ACM1, whereas failure to inhibit Cdh1 by Cdk phosphorylation is lethal.…”

Evolution of networks and sequences in eukaryotic cell cycle control

“…In animals, Emi1 is an E2F transcriptional target, which binds and inhibits Cdh1 [48]; in yeast, Acm1 similarly binds and inhibits Cdh1 [49] and is expressed specifically at G1-S, probably under SBF control [50]. However, Acm1 and Emi1 lack detectable sequence similarity [51]. Sic1 and Kip-Cip are both tight-binding stoichiometric inhibitors of cyclin-Cdk complexes, and Cdk1 phosphorylation targets both Sic1 and Kip for degradation via the SCF complexes ubiquitin ligase.…”