G1-faasi tsükliinist sõltuva kinaasi kompleksi Cln2-Cdk1 substraatide valiku mehhanismid

Saccharomyces cerevisiae Cdk1 reguleerib erinevate valkude fosforüleerimise kaudu kogu rakutsükli toimimist. Eri aegadel seondub Cdk1 erinevate tsükliinidega. Mitmed Cdk1 substraadid on enim fosforüleeritud G1-faasis, mil Cdk1 on kompleksis tsükliiniga Cln2. Käesolevas töös uuriti, mis mehhanismid võimaldavad Cln2-spetsiifilist fosforüleerimist. Esmalt leiti, et mitmete Cln2-Cdk1 substraatide fosforüleerimine ei vaja varem kirjeldatud LP-seondumist. Substraatide Sli15, Bud2 ja Rtt109 uurimisel selgus, et Cln2-Cdk1- vahendatud fosforüleerimist soodustavad lüsiini- või arginiinijääkide rikkad seondumismotiivid (KR-motiiv), millest tugevaima mõjuga on α-heeliksid, milles on vähemalt kolm positiivselt laetud aminohappejääki järjestikku heeliksi ühel küljel. Lisaks leiti Cln2 mutandid, millel on vähenenud spetsiifiline aktiivsus, ning näidati LP- ja KR-motiivide osalust polariseeritud kasvus. Veel näidati, et Cln2-Cdk1 reguleerib Sli15 paiknemist KRseondumise abil

Uurimus tsükliinist sõltuva kinaasi Cdk1 substraatide fosforüleerimise ajastamisest rakutsükli jooksul

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRakutsükkel on sündmuste jada, mis tagab rakkude jagunemise. Veatuks jagunemiseks peavad erinevad sündmused toimuma kindlas järjekorras ja sünkroniseeritult, nii et rakuline materjal esmalt kahekordistatakse ja seejärel jaotatakse kahe tütarraku vahel. Näiteks, paralleelselt DNA replikatsiooniga toimuvad ka tsentrosoomi duplikatsioon ja muutused raku metabolismis. Rakutsüklit koordineerivad tsükliinist sõltuvad kinaasid (CDK-d), mis fosforüleerimise kaudu reguleerivad sadade valkude aktiivsust. Kusjuures, muutused Cdk1 aktiivsuses on vajalikud ja piisavad, et käivitada DNA replikatsioon ja mitoos. CDK on katalüütiliselt aktiivne vaid kompleksis tsükliiniga ning erinevates rakutsükli faasides on avaldunud erinevad tsükliinid, mistõttu tekivad erinevad tsükliin-CDK kompleksid. CDK keskne roll rakutsüklis on pannud aluse küsimusele, millised mehhanismid võimaldavad sel kinaasil erineva ajastusega fosforüleerida sadu substraate. Selle küsimuse vastuseks on pakutud kaks mudelit. Rakutsükli jooksul CDK aktiivsus tõuseb ning kvantitatiivse mudeli kohaselt fosforüleerib CDK spetsiifilised valgud erinevatel kinaasi aktiivsuse tasemetel. Tsükliini spetsiifilisuse mudeli kohaselt aga suunavad erinevad tsükliinid CDK kompleksi kindlaid substraate fosforüleerima. Käesolevas töös uuriti Cdk1 kompleksi substraatide äratundmise mehhanisme eesmärgiga mõista, kuidas need interaktsioonid võimaldavad kinaasil Cdk1 erineva ajastusega sadu märklaudvalke fosforüleerida. Leiti, et substraatide fosforüleerimise ajastamisel on olulisel kohal nii CDK tõusev aktiivsus kui ka spetsiifilised tsükliin-substraat interaktsioonid. Lisaks kirjeldati uued tsükliin-substraat seondumismotiivid, mis on spetsiifilised kas S-, G2- või M-faasi tsükliinidele. Töös näidati, et erinevate motiivide, nagu fosforüleerimis- ja seondumismotiivide, mustrid määravad CDK substraatvalkude fosforüleerimise ajastuse rakutsüklis.Cell cycle is a series of events that guarantees the reproduction of cells. A flawless cell cycle requires a specific order of events, whereby the cellular contents are first duplicated and later segregated. Further, various events must be synchronized, for example DNA replication is coordinated with centrosome duplication and changes in cell metabolism. This is achieved by the action of cyclin-dependent kinases (CDKs) that phosphorylate and regulate the activity of hundreds of proteins. Interestingly, oscillation in the activity of Cdk1 is necessary and sufficient to initiate DNA replication and mitosis. CDKs require binding of cyclin proteins for enzymatic activity. Different waves of cyclins are expressed in G1, S, G2 and M phases, resulting in the formation of distinct cyclin-CDK complexes in different cell cycle stages. Two models have been proposed to answer the question of which mechanisms direct the timely phosphorylation of Cdk1 targets. The quantitative model states that specific proteins are phosphorylated at distinct CDK activity thresholds and thus the order of events is governed by the increase in kinase activity during the cell cycle. Alternatively, the cyclin specificity model proposes that different cyclins direct CDK to phosphorylate stage-specific substrates. In this study, the substrate targeting interactions of the Cdk1 complex were studied with the aim to understand the mechanisms that enable Cdk1 to differentially phosphorylate hundreds of proteins. We found that CDK thresholds can be encoded both based on the increase in CDK activity in the cell cycle and based on cyclin-specific substrate targeting. We identify novel cyclin docking motifs that enable specific phosphorylation by S, G2 or M phase CDK complexes. Importantly, we show that the pattern of linear motifs, including phosphorylation sites and docking motifs, can determine the timing of CDK substrate phosphorylation throughout the cell cycle.https://www.ester.ee/record=b541941

Fosforüleeritavate aminohapete paiknemise mõju Cdk1 substraatide multifosforüleerimise dünaamikale

2016-04-2

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression

Multisite phosphorylation networks as signal processors for Cdk1

The order and timing of cell cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDK). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that the cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, the distribution of serines and threonines as phospho-acceptors, and the positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phospho-adaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within the networks of phosphorylation sites in different targets provides a wide range of possibilities for the differential amplification of Cdk1 signals, providing a mechanism to generate a wide range of thresholds in the cell cycle