The Spo12 Protein of Saccharomyces cerevisiae: A Regulator of Mitotic Exit Whose Cell Cycle-Dependent Degradation Is Mediated by the Anaphase-Promoting Complex

Abstract The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis.

Download Full-text

The Ubiquitin-Ligase APC-Cdh1 Is Required for Maintaining Genomic Stability and Is Transcriptional Repressed by AML1/ETO.

Blood ◽

10.1182/blood.v106.11.1609.1609 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1609-1609

Author(s):

Ralph Waesch ◽

Dirk Engelbert ◽

Dominik Schnerch

Keyword(s):

Ubiquitin Ligase ◽

Transcriptional Repression ◽

Genetic Instability ◽

Kinase Inhibitor ◽

Cell Cycle Control ◽

Genomic Stability ◽

Cyclin B1 ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Cyclin Dependent Kinase Inhibitor

Abstract Accurate DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. We found that ubiquitin-dependent proteolytic cell cycle control by the E3-ubiquitin-ligase anaphase-promoting complex (APC) activated by Cdh1 (APC-Cdh1) is required for maintaining genomic integrity and viability in proliferating human cells. Lentiviral-delivered stable expression of short hairpins targeted against Cdh1 causes an apoptotic phenotype associated with p53-stabilization and p53-dependent transcriptional up-regulation of the cyclin-dependent kinase inhibitor p21. Cdh1-depleted cells enter mitosis delayed compared to controls suggesting the activation of a DNA-damage checkpoint in S- and G2-phase. Depletion of Cdh1 leads to premature accumulation of cyclin A2 and cyclin B1 in G1- and S-phase. This may interfere with loading of pre-replication-complexes onto origins of replication in G1 and cause chromosomal instability when cells progress into mitosis. In addition, stabilization of the Cdh1 target Aurora A at physiological levels is sufficient to cause centrosome overduplication and subsequent polyploidization in Cdh1 and p53 deficient cells. Genetic instability is a hallmark of cancer cells and deregulation of APC-Cdh1 may be involved, since we observe downregulation of Cdh1 in t(8;21) acute myeloid leukemia. Moreover, expression of AML1/ETO in an inducible system leads to downregulation of Cdh1 indicating transcriptional repression of Cdh1 by AML1/ETO. Consistent with these data AML1/ETO positive cell lines show numerical chromosomal aberrations. Thus, disruption of the central mitotic control machinery leads to genetic instability by several mechanisms and the APC may be an important suppressor of tumor progression in AML1/ETO positive leukemia.

Download Full-text

Uncovering the role of APC-Cdh1 in generating the dynamics of S-phase onset

Molecular Biology of the Cell ◽

10.1091/mbc.e13-08-0480 ◽

2014 ◽

Vol 25 (4) ◽

pp. 441-456 ◽

Cited By ~ 24

Author(s):

Xi Yuan ◽

Jeyaraman Srividhya ◽

Thomas De Luca ◽

Ju-hyong E. Lee ◽

Joseph R. Pomerening

Keyword(s):

Kinase Inhibitor ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Cyclin E1 ◽

Stimulus Response ◽

Cyclin Dependent Kinase Inhibitor ◽

E1 Protein ◽

Reduced Frequency ◽

Phase Entry

Cdh1, a coactivator of the anaphase-promoting complex (APC), is a potential tumor suppressor. Cdh1 ablation promotes precocious S-phase entry, but it was unclear how this affects DNA replication dynamics while contributing to genomic instability and tumorigenesis. We find that Cdh1 depletion causes early S-phase onset in conjunction with increase in Rb/E2F1-mediated cyclin E1 expression, but reduced levels of cyclin E1 protein promote this transition. We hypothesize that this is due to a weakened cyclin-dependent kinase inhibitor (CKI)–cyclin-dependent kinase 2 positive-feedback loop, normally generated by APC-Cdh1–mediated proteolysis of Skp2. Indeed, Cdh1 depletion increases Skp2 abundance while diminishing levels of the CKI p27. This lowers the level of cyclin E1 needed for S-phase entry and delays cyclin E1 proteolysis during S-phase progression while corresponding to slowed replication fork movement and reduced frequency of termination events. In summary, using both experimental and computational approaches, we show that APC-Cdh1 establishes a stimulus–response relationship that promotes S phase by ensuring that proper levels of p27 accumulate during G1 phase, and defects in its activation accelerate the timing of S-phase onset while prolonging its progression.

Download Full-text