Fission Yeast Receptor of Activated C Kinase (RACK1) Ortholog Cpc2 Regulates Mitotic Commitment through Wee1 Kinase

Abstract

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G 1 /S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G 2 /M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G 1 /S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.Cell reproduction involves the passing through a series of events collectively known as the cell cycle, which consists of alternative stages involving DNA replication (S phase), chromosome segregation and nuclear division (mitosis), as well as cell division (cytokinesis). Entry into mitosis is induced by the activation of a cyclin B-bound Cdc2/Cdk1 kinase, which is highly conserved among eukaryotic cells (1). In the fission yeast Schizosaccharomyces pombe, the inhibitory phosphorylation at a conserved tyrosine 15 (Tyr 15 ) in Cdc2 regulates its kinase activity that determines the mitotic onset and the start of division when the cells have reached a defined size (2). The kinase Wee1 down-regulates Cdc2 by inhibitory Tyr 15 phosphorylation, which is reversed by Cdc25 phosphatase, leading to Cdc2 activation and triggering of mitotic entry (3-6). Consequently, fission yeast cells lacking Wee1 activity enter mitosis before reaching a critical size to produce two small daughter cells (7). On the contrary, mutants in Cdc25 enter mitosis at an increased cell size, indicating that the activities/levels of both Cdc25 and Wee1 must be tightly regulated to provide an accurate control of the mitotic onset. In fission yeast, Wee1 is in turn phosphorylated and its activity negatively regulated by two SAD family kinases, Nim1 (also known as Cdr1 (8 -12) and Cdr2 (13,14). Recently, it has been described that gradients from the cell ends involving the DYRK family kinase Pom1 play a key role in the control of the progression of the cell cycle b...

The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast

“…Deletion of Cpc2p slightly increased the percentage of cells within a population contain multiple-septa (regardless of the presence or absence of Cyr1) and is consistent with previous reports [26]. Interestingly, and somewhat counter-intuitive, this effect was more pronounced upon overexpression of Cpc2p regardless of the presence (Wild type (JY448) = <0.1%; oe-cpc2 + (JY1711)  = 10±0.2% - p = <0.0001, n  = 4) or absence of Cyr1p (Wild type (JY546) = <0.1%; oe-cpc2 + (JY1578)  = 10±0.15% - p = <0.0001, n  = 4).…”

“…The fission yeast ortholog Cpc2p has been reported to modulate the stress-response pathway and positively regulates the G 2 /M transition at the ribosomal level [26]. Here we have described the role of Cpc2p in modulating the pheromone-response pathway.…”

“…Upon pheromone-stimulation activated Spk1p (the MAP kinase of the mating response) reduces Msa2p activity, allowing an increase in Ste11p translation [25]. Cpc2p appears to have a number of other cellular roles including; modulating the cell cycle of mitotically growing cells by regulating the G 2 /M transition [26] and a documented association with the 40S ribosomal subunit [27], that suggests a role in modulating the level of translation for many other genes.…”

A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

“…Cpc2 affects the efficient translation of a subset of proteins and may act as a scaffold for a number of signaling pathways in fission yeast 44,45 . In the absence of Cpc2 the level of Wee1 is increased, while the level of the Wee1 inhibitor Cdr2 is decreased, suggesting that the observed increased cell length at division of both the haploid gene deletion and diploid heterozygous gene deletion mutants could be due to a delay in activation of the Cdc2 kinase at the G2-M transition 46 . Cdr2 is a component of the Pom1 pathway and in our screen showed a statistically significant deviation in length at septation (+7.2%) to the control (Table S1B).…”

Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

“…We then examined whether the phosphorylation of Nrd1 affects its ability to bind to Cpc2, the RACK1 orthologue in fission yeast, since Cpc2 was identified as a binding partner for Nrd1 [13] and Cpc2 has roles in translation, G2/M transition, and stress responses in fission yeast [26], [27]. We co-expressed various versions of GST-fused Nrd1 proteins in cells expressing the chromosomally tagged Cpc2-GFP from its endogenous promoter.…”