Uurimustöö Saccharomyces cerevisiae tsükliinist sõltuva kinaasi Cdk1 substraadispetsiifilisusest ja multifosforüleerimise mehhanismist

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Raku jagunemistsükkel ehk rakutsükkel on protsess, mille käigus rakk kahekordistab oma sisu ja seejärel jaguneb kaheks. Rakutsükli saab jagada neljaks erinevaks etapiks: G1-, S-, G2- ja M-faasiks. Võtmetähtsusega sündmused – DNA replikatsioon ja kromosoomide segregatsioon ning järgnev tsütoplasma jagunemine ̶ toimuvad vastavalt S- ja M-faasis. S- ja M-faas on teineteisest eraldatud vaheetappide ehk G1- ja G2-faasiga. Rakutsükli faaside vaheldumine on reguleeritud kontrollsüsteemi poolt, mille peamisteks komponentideks on tsükliinist sõltuvad kinaasid (cyclin-dependent kinase; CDK). CDK valkude aktiivsuse ostsillatsioon sõltub erinevate regulatoorsete subühikute ehk tsükliinide olemasolust erinevates rakutsükli etappides. Tsükliinid võib jaotada kolme klassi: G1-tsükliinid, mis seonduvad CDK-dega G1-faasis, S-faasi tsükliinid, mis kontrollivad DNA replikatsiooni, ja mitootilised ehk B-tüüpi tsükliinid, mis aktiveerivad CDK-d rakutsükli G2- ja M-faasis. CDK-de ensümaatilist aktiivsus reguleeritakse nelja erineva mehhanismi abil: tsükliini seondumine, aktiveeriv või inhibeeriv fosforüleerimine ja seondumine inhibiitorvalkudega. Aktiivsed tsükliin-Cdk kompleksid toimivad lülititena, lisades teistele valkudele fosfaatrühmi ning muutes seeläbi nende omadusi. Enamus substraatvalke sisaldavad mitmeid CDK poolt äratuntavaid fosforüleerimise konsensusjärjestusi S/T-P-x-K/R (kus x võibolla ükskõik milline aminohape), milles aminohapped seriin (S) või treoniin (T) käituvad fosfaadi aktseptorina. Lisaks kuulub tsükliin-Cdk kompleksi veel CDK adaptorvalk Cks, moodustades kolmikkompleksi tsükliin-Cdk-Cks. Cks võib seonduda juba fosforüleeritud valkudega, aidates kaasa substraatide multi-fosforüleerimisele. Üldiselt määravad tsükliin-Cdk-Cks komplekside substraadi spetsiifilisust kolm äratundmismotiivi: 1) Tsükliinil asuv hüdrofoobne tasku, mis interakteerub substraatidel oleva seondumismotiiviga, 2) CDK aktiivsait, mis seondub sihtmärkvalgu konsensusjärjestusega ja 3) Cks-e katioonne tasku, mis seondub juba fosforüleeritud sihtmärkvalkude fosfaatrühma ja ümbritseva konsensusjärjestusega. Üheks mudelorganismiks, kus rakutsükli toimimismehhanisme uurida, on pagari¬pärm Saccharomyces cerevisiae. Erinevalt imetajatest leidub S. cere¬visieae-s ainult üks tsükliinist sõltuv kinaas, Cdk1, mis interakteerub erinevatel rakutsükli etappidel üheksa erineva tükliiniga (Cln1–3 ja Clb1–6) ning adaptor¬valgu Cks1-ga. Tsükliinid Cln1-3 on aktiivsed G1 faasis ja G1/S faasi üle¬minekul. Clb5 ja 6 vastutavad korrektse S-faasi sisenemise ja läbimise eest. Clb3 ja Clb4 osalevad G2/M üleminekul. Clb1 ja Clb2 aga kontrollivad mitoo¬tiliste rakkude saatust. Käesoleva eksperimentaalse töö esimene osa keskendub küsimusele, kuidas muutub erinevate tsükliin-Cdk1 komplekside aktiivsus S. cerevisiae rakutsükli käigus. Kuna enamus CDK sihtmärkvalkudest sisaldavad mitmeid fosforüleerimisjärjestusi, siis keskendusime eksperimentaalse töö teises osas multifosforüleerimise mehhanismi detailsele uurimisele CDK inhibiitorvalgu Sic1-e näitel. B-tüüpi tsükliinide Clb-Cdk1 komplekside inhibiitori Sic1-e tase hakkab tõusma mitoosi lõpus ja valk püsib aktiivsena hilise G1 faasini, kus toimub Sic1-e fosforüleerimisest sõltuv lagundamine. Spetsiifilistest lagundamisjärjestustest ehk degronitest fosforüleeritud Sic1 ära tundmine toimub läbi Cdc4, mis on ubikuitiini ligaasi SCF-i (Skp1/Cdc53/F-box) spetsiifilisusfaktor. Ubikuitineeritud Sic1-e lagundamine toimub üle proteasoomi raja. Oma töös uurisime põhjalikult erinevate tsükliin-Cdk1 komplekside poolt läbiviidavat Sic1 fosforüleerimist. Eksperimentaalse töö kolmandas osas uurisime erinevaid parameetreid, mis mõjutavad Cdk1 poolt läbiviidavat substraatvalkude multifosforüleerimist. Elemendid, mis määravad tsükliin-Cdk1-Cks1-st sõltuva multifosforüleerimise on järgmised: distantsid erinevate fosforüleerimisjärjestuste vahel, tsükliini seodumisjärjestuste positsioon fosforüleerimissaitide suhtes, Cks1 konsensusjärjestuse erinev spetsiifika, seriini- ja treoniinijääkide esinemise suhe CDK konsensusjärjestustes ja iga fosforüleerimisetapi protsessiivsusfaktor.The cell cycle is the process by which cells duplicate their contents and then divide to produce a pair of daughter cells. The master regulators of the cell cycle are cyclin dependent kinases (CDKs). CDKs are activated by their perio¬dically accumulating regulatory partners, the cyclins. The enzymatic activity of cyclin-Cdk complexes is tightly controlled by a variety of mechanisms. Sub¬strate targeting by a given cyclin-Cdk complex is mediated by the active site on the CDK and docking sites on the cyclin subunits. Additionally, the presence of a phosphate-binding pocket on the CDK adaptor subunit Cks1 promotes inter¬action with targets containing multiple phosphorylation sites. In simple euka¬ryotes, such as budding yeast, a single CDK, Cdk1, enzyme associates with se¬veral different cyclins. The combination of rising levels of CDK activity and the distinct substrate specificities of different cyclin-Cdk complexes enables the tem¬porally ordered phosphorylation of the many target proteins that regulate cell cycle events. Robust inhibition of S-phase CDK activity in the G1 phase of the cell cycle is the major mechanism preventing uncontrolled onset of DNA replication. In budding yeast, S phase is switched on after the rapid proteolytic degradation of the Cdk1 inhibitor Sic1. Sic1 is a stoichometric inhibitor of Clb-Cdk1 comple¬xes. It appears at the end of mitosis, and its destruction at the G1/S boundary is induced by Cdk1-mediated multisite phosphorylation. The first part of the present dissertation provides an overview of cell cycle control systems, focusing on the different substrate specificities of the various cyclin-Cdk complexes. Next, the CDK inhibitors in yeast and mammalian cells are introduced. Finally, the role of Cks1 as a phosphate binding adaptor mole¬cule for CDK, and the functional implications of this role are reviewed. The original results presented here cover the following areas: a) studies and discus-sions on the changes in cyclin-Cdk1 substrate specificity during the cell cycle b) in vivo and in vitro characterization and analysis of multisite phosphorylation of Sic1, and c) characterization of the parameters promoting Cks1-mediated multi-site phosphorylation of Cdk1 targets

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

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

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

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

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

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 Rbs 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

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

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

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 (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