rum1: a CDK inhibitor regulating G1 progression in fission yeast

In all eukaryotes, entry into mitosis from G2 phase is initiated by a complex of the cdc2 kinase and a B-type cyclin. It has now been shown that, in fission yeast, B-type cyclins also activate cdc2 in G1, thus governing cell-cycle commitment, as well as the onset of S phase. In this article, Karim Labib and Sergio Moreno review the evidence that ruml inhibits the kinase activity of cdc2 associated with B-type cyclins and is an important regulator o f G1 progression in fission yeast.     

Whi2 enhances methylmercury toxicity in yeast via inhibition of Akr1 palmitoyltransferase activity

BackgroundWe have previously reported that Whi2 enhances the toxicity of methylmercury in yeast. In the present study we examined the proteins known to interact with Whi2 to find those that influence the toxicity of methylmercury.MethodsGene disruption and site-directed mutagenesis were employed to examine the relationship of mercury toxicity and palmitoylation. Protein palmitoylation was examined using the acyl-biotinyl exchange method. Protein–protein interactions were detected by immunoprecipitation and immunoblotting.ResultsWe found that deletion of Akr1, a palmitoyltransferase, rendered yeast cells highly sensitive to methylmercury, and Akr1 is necessary for the methylmercury resistance of Whi2-deleted yeast. Palmitoyltransferase activity of Akr1 has an important role in the alleviation of methylmercury toxicity. Whi2 deletion or methylmercury treatment enhanced the palmitoyltransferase activity of Akr1, and methylmercury treatment reduced the binding between Akr1 and Whi2.ConclusionsWhi2 bonds to Akr1 (a protein that is able to alleviate methylmercury toxicity) and thus inhibits Akr1's palmitoyltransferase activity, which leads to enhanced methylmercury toxicity. In contrast, methylmercury might break the bond between Whi2 and Akr1, which enhances the palmitoyltransferase activity of Akr1 to alleviate methylmercury toxicity.General significanceThis study's findings propose that the Whi2/Akr1 system can be regarded as a defense mechanism that detects methylmercury incorporation of yeast cells and alleviates its toxicity.     

The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1)

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.     

Hexokinase 2 regulates G1/S checkpoint through CDK2 in cancer-associated fibroblasts

Hexokinase 2 (HK2), a pivotal glycolytic enzyme, is often overexpressed in tumor cells and contributes to glycolysis. Emerging evidence has suggested that glycolysis is also enhanced in cancer-associated fibroblasts (CAF). However, it is not clear whether HK2 is involved in enhanced glycolysis in CAFs or what role HK2 plays in the CAFs. In this study, both time course experiments and dose response experiments demonstrated that the protein and mRNA levels of HK2 increase in CAF cells, according to western blot and quantitative PCR analyses, respectively. Additionally, miR-182 targets the 3′ UTR of HK2, and its overexpression results in the degradation of HK2 mRNA, which eventually reduces the level of HK2 protein. On the other hand, knockdown of miR-182 increased the expression of HK2. Most importantly, HK2 regulated the protein level and T14 phosphorylation of CDK2, and knockdown of HK2 resulted in a G1 phase cell cycle arrest. These observations suggest that HK2 plays important roles in glycolysis regulation and in cell cycle checkpoint activation.     

IPP5, a novel protein inhibitor of protein phosphatase 1, promotes G1/S progression in a Thr-40-dependent manner

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here we describe the characterization of a novel inhibitory molecule for PP1, human inhibitor-5 of protein phosphatase 1 (IPP5). We find that IPP5, containing the PP1 inhibitory subunits, specifically interacts with the PP1 catalytic subunit and inhibits PP1 phosphatase activity. Furthermore, the mutation of Thr-40 within the inhibitory subunit of IPP5 into Ala eliminates the phosphorylation of IPP5 by protein kinase A and its inhibitor activity to PP1, whereas the mutation of Thr-40 within a truncated form of IPP5 into Asp can serve as a dominant active form of IPP5 in inhibiting PP1 activity. In IPP5-negative SW480 and IPP5-highly positive SW620 human colon cancer cells, we find that overexpression of IPP5 promotes the growth and accelerates the G(1)-S transition of SW480 cells in a Thr-40-dependent manner, which could be reversed by downregulation of the PP1 expression. Moreover, silencing of IPP5 inhibits the growth of SW620 cells both in vitro and in nude mice possibly by inducing G(0)/G(1) arrest but not by promoting apoptosis. According to its role in the promotion of cell cycle progression and cell growth, IPP5 up-regulates the expression of cyclin E and the phosphorylated form of retinoblastoma protein. Our findings suggest that IPP5, by acting as an inhibitory molecule for PP1, can promote tumor cell growth and cell cycle progression, and may be a promising target in cancer therapeutics in IPP5-highly expressing tumor cells.     

Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast.

Many eukaryotic cells arrest the cell cycle at G1 phase upon nutrient deprivation. In fission yeast, during nitrogen starvation, cells divide twice and arrest at G1. We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G2. Three loci (apc10, ste9/srw1 and rum1) were identified. The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation. apc10(+) is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins. Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex. A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC). A second gene, ste9(+)/srw1(+), encodes a member of the fizzy-related family, also regulators of the APC. Finally, Rum1 is a cyclin-dependent kinase (CDK) inhibitor which exists only in G1. The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G1.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.     

Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.