Cpc2, a Fission Yeast Homologue of Mammalian RACK1 Protein, Interacts with Ran1 (Pat1) Kinase To Regulate Cell Cycle Progression and Meiotic Development

Abstract

The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the ␤ subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2⌬ cells fail to accumulate in G 1 when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2⌬ cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.All living cells integrate signals from the environment to modify the activity of genes required for mitotic division, differentiation, or stationary phase. For the fission yeast Schizosaccharomyces pombe, nutritional signals direct life cycle choices. As key nutrients become limited, cells exit the mitotic cycle and enter either G 0 stationary phase or a program of sexual differentiation (6, 10). The choice between G 0 and differentiation is governed by the presence of mating-specific pheromones. Starved cells respond to pheromones produced by cells of the opposite mating type by undergoing transient G 1 arrest, followed by conjugation and meiosis (8,18,33,49). As expected from the need for both nutrient limitation and pheromone signaling, many signal transduction pathways converge to regulate differentiation. These include pathways regulated by cyclic AMP (cAMP), the ras1-regulated mitogen-activated protein (MAP) kinase pathway and the stress-activated MAP kinase pathway (see reference 51 for a review). However, a variety of studies indicate that each phase of the fission yeast life cycle can be governed by the activity of Ran1 (Pat1) kinase (referred to as Ran1 hereafter) and its substrates.Inactivation of Ran1 is necessary and sufficient to divert cells from the mitotic cell cycle into the meiotic developmental program (16,17,32). Experiments examining the phenotype of cells carrying a ran1 temperature-sensitive allele suggest that Ran1 is regulated by stepwise inactivation of the kinase (3, 23).Limiting nutritional conditions trigger partial inactivation ...

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

“…1A) (21,22). In eukaryotic cells, Cdk1/Cdc2 kinase activity is down-regulated during G 2 phase of the cell cycle by phosphorylation at Tyr 15 (2).…”

“…Also, genetic and biochemical approaches have shown that RACK1 negatively regulates cell cycle progression at the G 1 /S transition in mammalian cells by suppressing the activity of Src, a nonreceptor protein tyrosine kinase that plays a multitude of roles in cell signaling, and Src-mediated cell cycle regulators in G 1 , thereby promoting a delay of entry into S phase (29,30). However, fission yeast 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 (21). In this work we have taken advantage of S. pombe as a suitable model to identify molecular mechanisms related to cell cycle, and we show that Cpc2 is a key element involved in the regulation of the mitotic onset in this organism.…”

“…Further studies have shown its role as a scaffold which mediates cross-talk between various signaling pathways by binding in vivo to other proteins and regulating multiple biological processes like MAPK activation, angiogenesis, tumor growth, neuronal response, apoptosis, chromatin remodeling, and proper function of the circadian clock (17)(18)(19)(20). Cpc2, the fission yeast RACK1 ortholog, was identified through its action on protein kinase Ran1/Pat1, which controls the transition from mitosis to meiosis (21). Mutants lacking Cpc2 show several characteristic phenotypes like defective sexual differentiation under nitrogen deprivation and sensitivity to different stresses (21,22).…”

“…Cpc2, the fission yeast RACK1 ortholog, was identified through its action on protein kinase Ran1/Pat1, which controls the transition from mitosis to meiosis (21). Mutants lacking Cpc2 show several characteristic phenotypes like defective sexual differentiation under nitrogen deprivation and sensitivity to different stresses (21,22). Interestingly, RACK1/Cpc2 proteins are structural components of the 40 S ribosomal subunit (23)(24)(25)(26)(27), located in the proximity of the mRNA exit channel and in close contact with the binding surface of the eIF3 complex (27).…”

Function of p73, not of p53, is inhibited by the physical interaction with RACK1 and its inhibitory effect is counteracted by pRB

“…Our b-galactosidase assays showed that RACK1 at least in part inhibited the p73a-induced apoptosis. Recently, it has been reported that Cpc2, a yeast homolog of mammalian RACK1, has an important role for the cell cycle progression (Mcleod et al, 2000). As described by Zeng et al (2000), p300/CBP with HAT activity interacts with the NH 2 -terminus of p73, and stimulates both the transactivation and the proapoptotic function of p73.…”