Targets of the cyclin-dependent kinase Cdk1
TL;DR: The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylated of the molecular machines that drive cell-cycle events.
Abstract: The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.
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TL;DR: The components of the end resection machinery, the role of end structure, and the cell-cycle phase on resection and the interplay of end processing with NHEJ are reviewed.
Abstract: DNA double-strand breaks (DSBs) are cytotoxic lesions that can result in mutagenic events or cell death if left unrepaired or repaired inappropriately. Cells use two major pathways for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). The choice between these pathways depends on the phase of the cell cycle and the nature of the DSB ends. A critical determinant of repair pathway choice is the initiation of 5′-3′ resection of DNA ends, which commits cells to homology-dependent repair, and prevents repair by classical NHEJ. Here, we review the components of the end resection machinery, the role of end structure, and the cell-cycle phase on resection and the interplay of end processing with NHEJ.
1,363 citations
Cites background from "Targets of the cyclin-dependent kin..."
...Two other components of the resection apparatus, RPA and Dna2, are also CDK targets (43, 155)....
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TL;DR: A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of ∼700,000 potentially phosphorylatable residues.
Abstract: A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of approximately 700,000 potentially phosphorylatable residues. Multiple mechanisms have evolved that contribute to this exquisite specificity, including the structure of the catalytic site, local and distal interactions between the kinase and substrate, the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, systems-level competition between substrates, and error-correction mechanisms. The responsibility for the recognition of substrates by protein kinases appears to be distributed among a large number of independent, imperfect specificity mechanisms.
1,291 citations
Cites background from "Targets of the cyclin-dependent kin..."
...Systematic in vitro proteomic studies in Saccharomyces cerevisiae support this proposal: in one study, kinases phosphorylated between 1 and 256 substrates, and in a second study, cyclin-dependent kinase-1 ( Cdk1 ) from yeast phosphorylated hundreds of substrate...
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TL;DR: The role of cyclin-dependent kinases (Cdks) in regulating the mammalian cell cycle and their potential use as therapeutic targets in cancer has been investigated in this paper.
1,198 citations
TL;DR: The surprising redundancy amongst the classical cyclins, Cdk1 and Cdk2, and cyclin-dependent kinases show that the important differences between these proteins are when and where they are expressed rather than the proteins they phosphorylate.
1,105 citations
TL;DR: The successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins is reported using a prediction system for nuclear localization signals (NLSs) and the application of this strategy to other functional linear motifs should be useful in systematic studies of protein–protein networks.
Abstract: The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-α-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-α/β pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein–protein networks.
1,052 citations
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8,245 citations
TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd
5,301 citations
TL;DR: A comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle is created, and it is found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins.
Abstract: We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...
5,176 citations
TL;DR: Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport.
Abstract: Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport. Rab proteins and their effectors coordinate consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles to their target compartment. These molecules are highly compartmentalized in organelle membranes, making them excellent candidates for determining transport specificity and organelle identity.
3,373 citations
TL;DR: This work has shown that Cdk activity is governed by a complex network of regulatory subunits and phosphorylation events whose precise effects on Cdk conformation have been revealed by recent crystallographic studies.
Abstract: Cyclin-dependent kinases (Cdks) play a well-established role in the regulation of the eukaryotic cell division cycle and have also been implicated in the control of gene transcription and other processes. Cdk activity is governed by a complex network of regulatory subunits and phosphorylation events whose precise effects on Cdk conformation have been revealed by recent crystallographic studies. In the cell, these regulatory mechanisms generate an interlinked series of Cdk oscillators that trigger the events of cell division.
2,193 citations