Publications by authors named "Mardo Kõivomägi"

16 Publications

  • Page 1 of 1

Purification of Cyclin-Dependent Kinase Fusion Complexes for In Vitroh Analysis.

Methods Mol Biol 2021 ;2329:95-109

Department of Biology, Stanford University, Stanford, CA, USA.

Protein kinases are common elements in multiple signaling networks, influencing numerous downstream processes by directly phosphorylating specific target proteins. During the cell cycle, multiple complexes, each comprising one cyclin and one cyclin-dependent kinase (Cdk), function to regulate the orderly progression of cell cycle events. The mechanisms of cyclin-Cdk mediated control have, in part, been established through biochemical experiments involving the purification of cyclin and Cdk proteins to evaluate the activity of a given complex toward its target substrate proteins.Here I present a detailed procedure to simplify the preparation of cyclin-Cdk complexes by purifying them as a single fusion molecule with a 1:1 molar ratio and a detailed protocol for performing reconstituted kinases assays with the purified complexes.This methodology has allowed us to measure the activity and specificity of all budding yeast cyclin-Cdk1 complexes toward the model substrate histone H1. In addition, it has allowed us to perform kinase assays with a panel of purified human cyclin-Cdk complexes to analyze their specificity toward the retinoblastoma protein (Rb) and map the substrate cyclin-Cdk kinase docking interactions between Rb and human G1-Cdk complex.This chapter is focused on purification of cell cycle cyclin-Cdk complexes, but also affords a generalizable framework that can be adapted to other cyclin-dependent kinases like transcriptional cyclin-Cdks or any other multisubunit enzyme complexes. Taken together, the described workflow is a powerful and flexible biochemical platform for solving long-standing biological questions and has potential value in synthetic biology and in therapeutic discovery.
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http://dx.doi.org/10.1007/978-1-0716-1538-6_8DOI Listing
January 2021

Proline-Rich Motifs Control G2-CDK Target Phosphorylation and Priming an Anchoring Protein for Polo Kinase Localization.

Cell Rep 2020 06;31(11):107757

Institute of Technology, University of Tartu, Tartu 50411, Estonia. Electronic address:

The hydrophobic patch (hp), a docking pocket on cyclins of CDKs (cyclin-dependent kinases), has been thought to accommodate a single short linear motif (SLiM), the "RxL or Cy" docking motif. Here we show that hp can bind different motifs with high specificity. We identify a PxxPxF motif that is necessary for G2-cyclin Clb3 function in S. cerevisiae, and that mediates Clb3-Cdk1 phosphorylation of Ypr174c (proposed name: Cdc5 SPB anchor-Csa1) to regulate the localization of Polo kinase Cdc5. Similar motifs exist in other Clb3-Cdk1 targets. Our work completes the set of docking specificities for the four major cyclins: LP, RxL, PxxPxF, and LxF motifs for G1-, S-, G2-, and M-phase cyclins, respectively. Further, we show that variations in motifs can change their specificity for human cyclins. This diversity could provide complexity for the encoding of CDK thresholds to achieve ordered cell-cycle phosphorylation.
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http://dx.doi.org/10.1016/j.celrep.2020.107757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301157PMC
June 2020

A processive phosphorylation circuit with multiple kinase inputs and mutually diversional routes controls G1/S decision.

Nat Commun 2020 04 15;11(1):1836. Epub 2020 Apr 15.

Institute of Technology, University of Tartu, Tartu, 50411, Estonia.

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.
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http://dx.doi.org/10.1038/s41467-020-15685-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160111PMC
April 2020

Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

Mol Cell 2019 05 11;74(4):758-770.e4. Epub 2019 Apr 11.

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.
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http://dx.doi.org/10.1016/j.molcel.2019.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800134PMC
May 2019

A Precise Cdk Activity Threshold Determines Passage through the Restriction Point.

Mol Cell 2018 01;69(2):253-264.e5

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 that is passed when growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies have cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell-cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be assessed using a few critical protein activities.
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http://dx.doi.org/10.1016/j.molcel.2017.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790185PMC
January 2018

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase.

Cell Mol Life Sci 2017 09 28;74(18):3395-3412. Epub 2017 Apr 28.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, 84105, Beer-Sheva, Israel.

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.
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http://dx.doi.org/10.1007/s00018-017-2523-zDOI Listing
September 2017

Multistep phosphorylation systems: tunable components of biological signaling circuits.

Mol Biol Cell 2014 Nov;25(22):3456-60

Institute of Technology, University of Tartu, 50411 Tartu, Estonia

Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications.
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http://dx.doi.org/10.1091/mbc.E14-02-0774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4230602PMC
November 2014

Docking interactions: cell-cycle regulation and beyond.

Curr Biol 2014 Jul;24(14):R647-R649

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

In budding yeast, the mating pathway activates Far1 to inhibit G1 cyclins in complex with the cyclin-dependent kinase (Cln-Cdk). Yet, the molecular mechanism has remained largely unclear for over 20 years. A recent report helps shed light on this regulation.
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http://dx.doi.org/10.1016/j.cub.2014.05.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771416PMC
July 2014

Cks confers specificity to phosphorylation-dependent CDK signaling pathways.

Nat Struct Mol Biol 2013 Dec 3;20(12):1407-14. Epub 2013 Nov 3.

1] Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California, USA. [2].

Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (CDK) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical because Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding-yeast Cks associates with select phosphorylated sequences in cell cycle-regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances CDK activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed CDK substrates and binding partners. We characterize new Cks-binding sites in the mitotic regulator Wee1 and discover a new role for Cks in regulating CDK activity at mitotic entry. Together, our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for CDK activity.
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http://dx.doi.org/10.1038/nsmb.2707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242096PMC
December 2013

Multisite phosphorylation networks as signal processors for Cdk1.

Nat Struct Mol Biol 2013 Dec 3;20(12):1415-24. Epub 2013 Nov 3.

Institute of Technology, University of Tartu, Tartu, Estonia.

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.
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http://dx.doi.org/10.1038/nsmb.2706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855452PMC
December 2013

Double-negative feedback between S-phase cyclin-CDK and CKI generates abruptness in the G1/S switch.

Front Physiol 2012 6;3:459. Epub 2012 Dec 6.

Institute of Technology, University of Tartu Tartu, Estonia.

The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high cyclin-dependent kinases (CDK) inhibitor (CKI) levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK (Cln1,2-Cdk1) and S-phase CDK (Clb5,6-Cdk1) complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double-negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex. In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.
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http://dx.doi.org/10.3389/fphys.2012.00459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515773PMC
December 2012

Cdk1: a kinase with changing substrate specificity.

Cell Cycle 2011 Nov 1;10(21):3625-6. Epub 2011 Nov 1.

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http://dx.doi.org/10.4161/cc.10.21.17854DOI Listing
November 2011

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase.

Nature 2011 Oct 12;480(7375):128-31. Epub 2011 Oct 12.

Institute of Technology, University of Tartu, Tartu 50411, Estonia.

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.
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http://dx.doi.org/10.1038/nature10560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228899PMC
October 2011

Dynamics of Cdk1 substrate specificity during the cell cycle.

Mol Cell 2011 Jun;42(5):610-23

Institute of Technology, University of Tartu, Tartu 50411, Estonia.

Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
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http://dx.doi.org/10.1016/j.molcel.2011.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115021PMC
June 2011

Phospho-regulation of kinesin-5 during anaphase spindle elongation.

J Cell Sci 2011 Mar;124(Pt 6):873-8

Department of Clinical Biochemistry, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel.

The kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.
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http://dx.doi.org/10.1242/jcs.077396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048887PMC
March 2011

Human TRB3 is upregulated in stressed cells by the induction of translationally efficient mRNA containing a truncated 5'-UTR.

Gene 2009 Sep 6;444(1-2):24-32. Epub 2009 Jun 6.

Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia.

Tribbles homolog 3 (TRB3) is a pseudokinase that has been implicated in the control of stress response, cell viability and metabolic processes, and has been linked to medical conditions, including insulin resistance, cardiovascular disease and diabetes. Therefore, the understanding of mechanisms that regulate TRB3 expression is of considerable importance. We have previously described the existence of several human (h) TRB3 mRNA isoforms that differ in their 5'-untranslated region (5'-UTR). In this study, we use a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system to characterize the expression levels of hTRB3 mRNA isoforms in HepG2 hepatoma cells cultured in regular medium or exposed to arsenite, and investigate the effect of hTRB3 5'-UTR variants on the efficiency of mRNA translation. The data indicate that of the hTRB3 mRNA splice variants, 1A is predominant (>80% of molecules) in both the stressed and unstressed states, and that the remainder consists mainly of 1B4, with the variants 1B1, 1B2 and 1B3 together forming less than 1% of the population in either condition. In addition to the substantial transcriptional upregulation of all hTRB3 mRNA splice variants, the exposure of cells to arsenite results in a marked increase in the proportion of splice variant 1A molecules containing a truncated 5'-UTR. The shortened 1A 5'-UTR proved to be translationally more efficient than the untruncated 1A 5'-UTR, due to the lack of an inhibitory upstream open reading frame (uORF). Thus, increased transcription as well as altered usage of 5'-UTR variants contributes to the upregulation of hTRB3 protein synthesis in stressful conditions.
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http://dx.doi.org/10.1016/j.gene.2009.06.001DOI Listing
September 2009