Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

CDK Regulation of Meiosis: Lessons from S. cerevisiae and S. pombe

Meiotic progression requires precise orchestration, such that one round of DNA replication is followed by two meiotic divisions. The order and timing of meiotic events is controlled through the modulation of the phosphorylation state of proteins. Key components of this phospho-regulatory system include cyclin-dependent kinase (CDK) and its cyclin regulatory subunits. Over the past two decades, studies in budding and fission yeast have greatly informed our understanding of the role of CDK in meiotic regulation. In this review, we provide an overview of how CDK controls meiotic events in both budding and fission yeast. We discuss mechanisms of CDK regulation through post-translational modifications and changes in the levels of cyclins. Finally, we highlight the similarities and differences in CDK regulation between the two yeast species. Since CDK and many meiotic regulators are highly conserved, the findings in budding and fission yeasts have revealed conserved mechanisms of meiotic regulation among eukaryotes.

Comprehensive analysis of fungal G1 cyclin docking motif sequences that control CDK regulatory potency in vivo

SUMMARYCyclin-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.

Interchangeable Roles for E2F Transcriptional Repression by the Retinoblastoma Protein and p27KIP1–Cyclin-Dependent Kinase Regulation in Cell Cycle Control and Tumor Suppression

ABSTRACT The mammalian G1-S phase transition is controlled by the opposing forces of cyclin-dependent kinases (CDK) and the retinoblastoma protein (pRB). Here, we present evidence for systems-level control of cell cycle arrest by pRB-E2F and p27-CDK regulation. By introducing a point mutant allele of pRB that is defective for E2F repression (Rb1 G ) into a p27KIP1 null background (Cdkn1b −/−), both E2F transcriptional repression and CDK regulation are compromised. These double-mutant Rb1 G/G ; Cdkn1b −/− mice are viable and phenocopy Rb1 +/− mice in developing pituitary adenocarcinomas, even though neither single mutant strain is cancer prone. Combined loss of pRB-E2F transcriptional regulation and p27KIP1 leads to defective proliferative control in response to various types of DNA damage. In addition, Rb1 G/G ; Cdkn1b −/− fibroblasts immortalize faster in culture and more frequently than either single mutant genotype. Importantly, the synthetic DNA damage arrest defect caused by Rb1 G/G ; Cdkn1b −/− mutations is evident in the developing intermediate pituitary lobe where tumors ultimately arise. Our work identifies a unique relationship between pRB-E2F and p27-CDK control and offers in vivo evidence that pRB is capable of cell cycle control through E2F-independent effects.

Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

CDK Phosphorylation of a Novel NLS-NES Module Distributed between Two Subunits of the Mcm2-7 Complex Prevents Chromosomal Rereplication

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.