The microtubule depolymerase MCAK influences chromosomal instability (CIN), but what controls its activity remains unclear. Bendre et al. show that GTSE1, a protein found overexpressed in tumors, regulates microtubule stability and chromosome alignment during mitosis by inhibiting MCAK. High levels of GTSE1 are linked to chromosome missegregation and CIN.
Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin-Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin-Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3-Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.in vivo enzyme complexes screen | cyclin specificity A central problem in biology is determining the functions of individual enzyme subunits and the synergistic relationships among them. For example, enzymes that perform posttranslational modifications on proteins, such as the ubiquitin ligases, protein kinases and protein phosphatases, require different subunits to perform multiple transfer steps, assure specific subcellular localization, or provide additional specificity to substrate recognition (1-4). Cyclin-dependent kinases (Cdks) are a case in point, and the budding yeast Saccharomyces cerevisiae Cdk1 (also called Cdc28) is a very well-studied example of an enzyme of this category (5). Cdk1 requires the association of one of nine available cyclin partner proteins to recognize and phosphorylate its substrates (6, 7). The different Cdk1-cyclin complexes play critical roles in orchestrating the temporal and spatial ordering of events from initiation of the G1 transcriptional program (Cln1, -2, and -3) to DNA replication (Clb5 and -6), spindle assembly (Clb3 and -4), and mitosis (Clb1 and -2) (8).The crucial role of Cdk1 in cell cycle regulation has prompted several extensive or proteome-wide studies to identify Cdk1 substrates or cyclin targets (9-12). To date, no experimental approach has captured interactions between Cdk1 and its substrates and the dependency of this interaction on one or more cyclins in the context of a living cell. In this study, we describe an approach that captures direct interactions between Cdk1 and its substrates and reveals the dependency of this interaction on one or more cyclins in living cells.We devised a simple in vivo screening strategy to both identify potential Cdk1 substrates and establish dependencies of the Cdk1 interactions with these substrates on specific cyclins using the optimized yeast S. cerevisiae prodrug-converting enzyme cytosine deaminase protein-fragment complementation assay (OyCD PCA...
Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.
Kinesin-5 proteins are microtubule associated motors, which are highly conserved from yeast to human cells. They share high homology in their catalytic motor domain sequence, fulfill similar essential mitotic roles in spindle assembly and anaphase B spindle elongation and, until recently (Roostalu et al., Science, 2011), were all thought to move towards plus ends of microtubules. Mechanisms that regulate Kinesin-5 function, specifically during anaphase B, are not well understood. S. cerevisiae cells express two Kinesin-5 homologues, Cin8 and Kip1, which overlap in function. Here we have examined in vitro and in vivo functions and regulation of Cin8 during anaphase B. We followed Cin8 localization and carried out single molecule fluorescence motility assays to study Cin8 motile properties. We found that in vitro, Cin8 molecules are able to switch directionality along a single microtubule as a function of ionic strength conditions and that during anaphase B, Cin8 moves not only towards the plus, but also towards the minus ends of spindle microtubules. Compared to kinesin-5 homologues of higher eukaryotes, S. cerevisiae Cin8 carries a uniquely large insert in loop 8 in its motor domain. To probe the role of the large loop 8 in the directionality switch of Cin8, we studied a construct in which this segment was replaced with the seven amino acids of loop 8 in the related S. cerevisiae kinesin-5 Kip1 (Cin8D99) (Hoyt et al.,J Cell Biol, 1992). We examined the anaphase B localization and in vitro motile properties of the Cin8D99 variant. Using combined in vitro and in vivo approaches, we were able to characterize the role of loop 8 in controlling Cin8 motility and function during S. cerevisiae anaphase.
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