Cdc28 and Cdc14 Control Stability of the Anaphase-promoting Complex Inhibitor Acm1

Abstract

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC Cdh1 inhibitor. Acm1 disappears in late mitosis when APC Cdh1 becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G 1 /S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.

Cdc14p resets the competency of replication licensing by dephosphorylating multiple initiation proteins during mitotic exit in budding yeast

“…C283S has previously been used as a substrate trap to capture the complexes formed between Cdc14p and its cognate phospho-substrates (Hall et al, 2008). We found that Orc2p, Orc6p and Mcm3p could be co-immunoprecipitated with FLAGCdc14 C283S , but not wild-type FLAG-Cdc14p, expressed from the GAL1 promoter to override the sequestration of Cdc14p by its inhibitors in mitotic cells (Fig.…”

“…We then used a well-established in vitro Cdc14p phosphatase assay (Visintin et al, 1998;Jaspersen and Morgan, 2000;Pereira et al, 2002;Bloom and Cross, 2007;Hall et al, 2008;ClementeBlanco et al, 2009) to test whether Cdc14p is able to dephosphorylate the initiation proteins in vitro. Orc2p, Orc6p, Cdc6p and Mcm3p isolated from mitotic cells were efficiently dephosphorylated in vitro when they were incubated with recombinant GST-tagged Cdc14 (GST-Cdc14) (Fig.…”

“…All pRS416-GAL1-3FLAG-based plasmids were derived from pHLP112 (Hall et al, 2008) by inserting the corresponding open reading frames (ORFs) between the NotI and XhoI sites to replace ACM1. All wild-type ORF inserts were generated by PCR amplification with KOD polymerase (Toyobo, Osaka, Japan) using W303-1a genomic DNA as the template.…”

Acm1 contributes to nuclear positioning by inhibiting Cdh1-substrate interactions

“…36 The degradation of Swe1 itself may be at least partially APC-dependent. 37 Thus, one possibility is that following Acm1 degradation initiated in early anaphase, 8,9,12 Cdh1 is recruited to the bud neck via the high affinity interaction with its substrate Hsl1. This specific localization could then allow the rapid, efficient and coordinated clearance of several key Cdh1 substrates once APC Cdh1 is activated by the mitotic exit network and Cdc14-catalyzed Cdh1 dephosphorylation.…”

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

“…We conclude that Cdc28 likely regulates Acm1 stability by direct phosphorylation and that these four phosphorylation sites have additive effects on Acm1 stability in vivo. While this report was under review, Hall et al also reported a role for Thr-161 phosphorylation in stabilizing Acm1 (26), though they did not examine the other single-point mutations.…”