Mitogen-activated protein kinases (MAPKs) play vital roles in multiple cellular processes and represent prominently pursued targets for development of therapeutic regimes. The MAPK Spc1 (p38 homologue) is known to be very important for both mitotic promotion and delay in Schizosaccharomyces pombe. However, the mechanism responsible for mitotic inhibition has remained elusive. Cdc25 (Cdc2 activator) and Wee1 (Cdc2 inhibtor) are important determinants of mitotic timing in all eukaryotes. Our results show that Spc1 can sense the perturbations in the balance of Cdc25 and Wee1 activities in S. pombe and that its function as a mitotic inhibitor is very important for controlling the same. An Spc1-Srk1-Rad24-dependent pathway for mitotic inhibition has been reported earlier.Here we report the presence of an alternative mechanism wherein Spc1 targets the 14-3-3 protein, Rad24, independently of Srk1, leading to relocalization of Cdc25 and mitotic inhibition. Our observations suggest that this pathway can serve as a backup mechanism for Cdc2 inactivation in the absence of Wee1.
Cyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation..
Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G 2 /M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G 2 /M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G 2 /M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G 2 /M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G 2 /M transition. The mammalian basic leucine zipper domain (bZIP) family transcription factor ATF2 is known to be associated with multiple cellular processes, including stress responses, DNA damage responses, and cell cycle regulation. Schizosaccharomyces pombe has a well-characterized ATF2 homolog (Atf1) with functions similar to those of the human ATF2 protein (1-4). It is important for heterochromatin formation and meiotic recombination. Atf1 has also been shown to influence some very important events during S. pombe cell division. In S. pombe, Atf1 was first isolated as the suppressor of the ⌬spc1 phenotype (1). Spc1 is the major mitogenactivated protein kinase (MAPK) in S. pombe and is the homolog of mammalian p38MAPK. It has also been implicated at many important stages of cell cycle control in S. pombe. Atf1 is known to be associated with activation of the spindle orientation checkpoint (5) that controls the metaphase-to-anaphase transition and activation of the anaphase-promoting complex (APC) leading to mitotic exit. It has a synthetic lethal interaction with Cut1 (6). Atf1 is also necessary for accumulation of cells in G 1 after nitrogen starvation (1). It has been shown to be important for degradation of the mitotic cyclin Cdc13 by activating the APC/cyclosome (APC/C) ubiquitin ligase (7).The major point of regulation of the S. pombe cell cycle is the transition from G 2 phase into mitosis. This transition is dependent on the activity of the cyclin-dependent kinase (CDK) Cdc2. The known important regulators of Cdc2 activity in S. pombe a...
The mechanism underlying stringently controlled sequence of events in the eukaryotic cell cycle involves periodic transcription of a number of genes encoding important regulators of cell cycle, growth, proliferation and apoptosis. Deregulated activities of transcription factors that contribute to this programmed gene expression, are associated with many diseases including cancer. A detailed mechanistic understanding of the transcriptional control associated with cell division is, therefore, important. We have reported earlier that the transcription factor Atf1 in Schizosaccharomyces pombe can regulate G2–M transition by directly controlling the expression of the mitotic cyclin Cdc13 (1).To gain a better understanding of the role of Atf1 in cell cycle, we performed a microarray based identification of cell cycle related targets of Atf1. The microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series (accession number GSE71820). Here we report the annotation of the genes whose expression get altered by Atf1 overexpression and also provide details related to sample processing and statistical analysis of our microarray data.
The transcription factor Atf1 is known to promote cell survival during various stress conditions in Schizosaccharomyces pombe by activating the expression of appropriate genes. It can also activate transcription of other important genes responsible for cell cycle progression. An Atf1-dependent increase in the expression of cell division promoting genes will oppose activation of checkpoints necessary to ensure repairs and cell survival during stress. Hence, selective inhibition of the cell cycle-related functions of Atf1 would be indispensable for cellular survival during stress. Here we present evidence in favour of selective inhibition of Atf1's ability to activate cdc13+ transcription. We show that the transcription factor Pcr1 can specifically inhibit the recruitment of Atf1 on cdc13 promoter and thereby prevent Atf1-mediated mitotic acceleration. We also show that this opposition of Atf1 functions by Pcr1 extends to the G1-S transition event as well. Altogether these results suggest a previously unknown antagonistic function of Atf1 and Pcr1 in regulating Cdc13 expression during cell cycle progression.
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