The roles of cyclin-dependent kinases (Cdks) in regulation of transcription and cell cycle Dalibor Blazek Transcriptional regulation group Molecular Medicine CEITEC-MU Human kinases Cyclin-dependent kinases (Cdks) Cyclin-dependent kinases (Cdks) Protein complexes that compose of 1) Kinase subunit 2) Cyclin subunit Serine-threonine kinases-regulate function of proteins by phosphorylation of either Serine (S) or Threonine (T) Sequence preference motif: S/T-P-X-K/R Both subunits needed for the kinase activity of the complex Most Cdks usually have at least one Cyclin partner In humans there are at least 21 genes encoding Cdks however only about half of the Cdks are sufficiently studied = relatively well studied Cdks Human cell has 21 Cdks and 29 Cyclins The Cdk complexes regulate various processes in cells Major functions: -Regulation of Cell Cycle (Cdk1,2,4,6,7) -Regulation of Transcription (Cdk7,8,9,12) Other functions: - regulation of pre-mRNA processing (Cdk11, Cdk9) - regulation of neuronal cell differentiation (Cdk5) - likely more functions to be discovered Cdk complexes regulate various processes in cells Regulation of kinase activity of Cdk complexes-overview Activation of Cdk kinase activity: -Association of Cdk with various Cyclin subunits -Phosphorylation of threonine in the “T-loop” of Cdk -Degradation of Cdk inhibitor proteins by ubiqitination and proteolysis Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins to Cyc/Cdk complexes -Inhibitory phosphorylation of Cdk -Ubiqitination and degradation of Cyclins in proteasome -Binding of Cdk inhibitor proteins together with small nuclear RNA to Cyc/Cdk complex Activation of Cdk kinase activity: -Association of Cdk with various Cyclin subunits -Phosphorylation of Threonine in the “T-loop” of Cdk T-loop blocks active site (active site=ATP binding site) T-loop moves out of the active site P-T-loop improves binding of substrate Activation of Cdk kinase activity-Cdk2-Cyclin A Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins to Cyc/Cdk complexes P27 binding distorts and binds into the active site of Cdk2 (for example inhibits G1/S-Cdk in G1 phase) Activation of Cdk kinase activity: -Degradation of Cdk inhibitor proteins by ubiqitination and proteolysis Cell cycle-dependent phosphorylation of Cdk inhibitor is a “mark” for recognition by SCF ubiquitin ligase, ubiquitinylation and degradation, rendering Cyc/Cdk complex more active Inhibition of Cdk kinase activity: -Inhibitory phosphorylation of Cdk Inhibition of Cdk kinase activity: -Ubiquitination and degradation of Cyclin by proteasome Mitosis-dependent activation of APC ubiquitin ligase leads to ubiquitination of Cyclin and its degradation Inhibition of Cdk kinase activity: -Binding of Cdk inhibitor proteins and 7SK small nuclear RNA (7SK snRNA ) to CycT/Cdk9 complex P-TEFb=Cdk9 The kinase activity of Cdk9 is inhibited by binding to several proteins and small nuclear RNA, 7SK snRNA Regulation of Cell Cycle by Cdks Cell Cycle Cell cycle leads to production of two genetically identical daughter cells Major events of the cell cycle S-phase – DNA synthesis-duplication of the chromosomes M-phase – mitosis-pair of chromosomes segregated into the nuclei – cytokinesis- the cell divides into two identical cells The cell cycle has four phases G1 and G2 phases-time delay to allow the growth of the cell -time to monitor external and internal conditions before commitment to onset of S and M phase The control of the cell cycle-three major checkpoints Control of the cell cycle triggers essential processes such as DNA replication, mitosis and cytogenesis Cell cycle control system depends on cyclically activated Cdks Cyclical changes (expression and degradation) in Cyclin protein levels result in cyclic assembly/disassembly and activation/inhibition of Cyc/Cdk complexes; this leads to phosphorylation/dephosphorylation of proteins that initiate and regulate cell cycle events Cyclin protein levels change, Cdk protein levels are constant Major Cyclins and Cdks in Vertebrates and Yeast Comparison of the yeast and mammalian cell cycle Yeast- cell cycle is directed by one Cdk-Cdk1 (cdc28) Mammals-several Cdks (classical model), Cdk1 is essential to drive cell cycle in the absence of other Cdk (mouse knock out model) Evolution of cell cycle control Cell cycle control system is a network of biochemical switches where Cyc/Cdk complexes play a major role Cyc/Cdk: Cell cycle phases: Event: Activation of M-Cdk (cycB/cdk1) De-phosphorylation activates accumulated M-Cdk at the onset of mitosis End of G2 Mechanism of cell cycle arrest in G1 by DNA damage DNA damage causes transcription of p21, Cdk inhibitory protein, that inhibits G1-S- and S-Cdks, arresting the cell cycle in G1 phase Deregulation of cell cycle and cancer Cells escape from the proper control of the cell cycle during cancer development: -Increase in expression and activity of proteins driving cell cycle regulators (Cdks) -Inactivation of inhibitors of Cdks Regulation of transcription by Cdks Transcriptional Cyc/Cdk complexes Major differences between Transcription and Cell Cycle Cyc/Cdk complexes Trancription Cyc/Cdks complexes: 1)Cdk has usually only one Cyclin partner 2)Usually in multi-protein complexes 3)The Cyclin levels in cells do not oscilate (Cdks need to be constantly active for basal transcription) 4)Regulated at the level of recruitment to specific gene Ad 4) Examples of recruitment of P-TEFb (Cdk9) to genes Differences between Cell Cycle and Transcription Cyc/Cdks-structure Sparse number of contacts btw Cyc and Cdk in transcription Cyc/Cdk complexes More contacts in Cell Cycle Cyc/Cdk complexes - important for Cdk activation Differences between Cell Cycle and Transcription Cyc/Cdks- Cyclin structure All Cyclins have 2 canonical cyclin-boxes responsible for Cdk binding Each cyclin-box consists of 5 helixes The cyclin-boxes conserved in all Cyclins Cell Cycle and Transcription Cyclins differ significantly in sequence and structure outside of the cyclin boxes (binding to other proteins) Differences between Cell Cycle and Transcription Cyc/Cdks- Cyclin structure Comparison of Cdk9 and Cdk2 Structures very similar, sequence similarity 40% Cdk9 (green) /Cdk2 (orange) T-loop (T186/T180) Transcription (Gene expression) Transcription- synthesis of RNA from DNA template Transcription in eukaryotes is tightly linked to cotranscriptional mRNA processing The co-transcriptional mRNA processing (capping, splicing, 3` prime end processing) Transcription of protein-coding genes by RNA polymerase II (RNAPII) Promoter RNAPII RNAPII RNAPII Initiation Elongation Termination AAAA mRNA GenePromoter RNAPII Pre-initiating RNAPII CTD CTD CTD CTD C-terminal domain (CTD) of RNAPII plays a crucial role in regulation of transcription and co-transcriptional mRNA-processing Pre-mRNA CTD consists of 52 repeats of heptapeptide YSPTSPS in which individual amino acids get phosphorylated to form a “CTD code” (Y1-S2-P3-T4-S5-P6-S7)x52 P P P PP RNAPII CTD iso iso -52 repeats in humans (21 consensus, 31 non-consensus) -26 repeats in yeast -evolutionary conserved-important! Human “CTD code” RNAPII Repeats of the CTD get phosphorylated by the Cdks (Y1-S2-P3-T4-S5-P6-S7)x52 Cdk9 Cdk7 P P P PP iso iso Cdk9 phosphorylates Serine (Ser) in the position 2 Cdk7 phosphorylates Serine (Ser) in the position 5 For the regulation of transcription cycle the phosphorylations of the CTD by the Cyc/Cdks are essential Promoter RNAPII GTFs Cdk7 Promoter RNAPII GTFs RNAPII Cdk9 RNAPII RNAPII Pre-initiating RNAPII Initiated RNAPII Elongation Termination Ser2P AAAA Ser5P Mediator Capping enzyme Splicing/Chromatin remodeling Cleavage/PolyA factors factors Modified CTD is a binding platform for transcription factors, RNA-processing factors and histone modification factors (code readers) RNAPII CTD Transcription factors RNA-processing factors Histone modification factors Exon Pre-mRNA Histones Phosphorylation of the CTD mediates: Transcription mRNA-processing Chromatin modifications RNA export Transcription-coupled genome stability CTD code readers Distribution of phosphorylated Serine 5 and Serine 2 in the CTD of RNAPII along the human protein coding genes Cdk7 (initiation) Cdk9 (elongation) RNAPII (Total) RNAPII Ser5-P RNAPII Ser2-P gene Roles of new Cdks in the CTD modification (CTD code) (Y1-S2-P3-T4-S5-P6-S7)x52 Cdk9 Cdk7 P P P PP iso iso Cdk12 Cdk13 ? Cdk7Cdk9 ? Cdks and their roles in transcriptional cycle of yeast and human Deregulation of transcription by Cdks leads to the onset of human diseases -Cancer - aberrant kinase activity of Cdk9 , Cdk12 defective transcriptional elongation, mRNA processing -HIV transcription- HIV Tat protein “steals” Cdk9 from its cellular complex to transcribe HIV genome Cdk9 is recruited to most of RNAPII promoters and is present in catalytically active (small) and inactive (large) complexes and regulates transcriptional elongation MEPCE LARP7 Cdk9-dependent transcriptional elongation is a highly regulated process and its deregulation can lead to the onset of cancer HOX genes MYC gene Cdk9 Cdk9 Brd4 2- 2Mixed Lineage Leukemia (MLL) Abnormal fusion of MLL protein with Cdk9-containing complexes leads to aberrant elongation of Hox genes in leukemic cells Acute Myeloid Leukemia (AML) Expression of Myc gene regulated at the level of Cdk9-dependent transcriptional elongation in this Myc-dependent cancer. Cdk12 is one of the most often mutated genes in ovarian carcinoma KD=kinase domain The mutations probably lead to the aberrant kinase activity and defective transcriptional elongation and/or mRNA processing of certain genes Cdk12 proposed to be a novel tumor suppressor W719del E928fs R882L E901C G909R K975E L996L T1014del HIV transcription is dependent on the Cdk9 (P-TEFb) protein HIV Tat protein “steals” Cdk9 from its complex with inhibitory Hexim1/7SK snRNA; resulting Tat/Cdk9 complex binds to HIV -TAR RNA element and drives HIV transcription in human cells Regulation of transcription (gene expression) by cyclin-dependent kinases Cyclin K/Cdk12-an emerging player in the transcription-coupled genome stability Role of Cyclin K/Cdk12 in the onset and maintenance of ovarian cancer Historically, Cdk9 and one of the cyclins (CycT1, CycT2 and CycK) were thought to form positive transcription elongation factor b (P-TEFb)-situation in 2008 Cdk9 Cyclin Cdk9 Cdk9 Cdk9 CyclinT1 CyclinT2 CyclinK P-TEFb Mostly studied Supports HIV transcription ? Not studied Does not support HIV transcription ? CycK binds Cdk12 and Cdk13 in two separate complexes: CycK/Cdk12 and CycK/Cdk13 Blazek et al, G&D, 2011 Cdk13 CycK Cdk12 CycK Kohoutek and Blazek, Cell Div. 2012 Cdk12 and Cdk13 proteins have similar kinase domains (similarity 93%), but the other domains are different cyclin box 1 cyclin box 2 proline rich motifCycK 1-580 44-152 160-258 320 568 Cyclin-dependent kinase (cdk) family (according to similarity of kinase domains) Adapted according to Malumbres et al., Nature Cell Biol., 2009 +CyclinT1/CyclinT2 = P-TEFb +CyclinK Cdk12 is a transcription-associated kinase phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII) (Y1-S2-P3-T4-S5-P6-S7)x52 P RNAPII CTD PP P P Cdk12 Transcriptional cyclin-dependent kinases phosphorylate the Cterminal domain (CTD) of RNA Polymerase II (RNAPII) and other factors to regulate individual steps of transcription Promoter RNAPII Cdk7 Promoter RNAPII RNAPII Cdk9 RNAPII RNAPII PIC recruitment (initiation) Promoter escape and abortive transcription Elongation Termination Ser5P Ser5P Ser2P AAAAA Initiation Elongation Termination Cdk12 ? CTD CTD CTD CTD mRNA Depletion of CycK/Cdk12 decreases the expression of a small subset of genes Blazek et al, G&D, 2011 Depletion of CycK/Cdk12 changes the expression of crucial DNA damage response genes Blazek et al, G&D, 2011 DNA content DNA content DNA content Numberofcells Numberofcells Numberofcells siRNA C siRNA CycK siRNA Cdk12 ……and depletion of CycK/Cdk12 leads to accumulation of cells in G2/M phase of cell cycle BRCA1, Fanconi anemia proteins, ATR-guardians of genome stability Friedenson, BMC cancer 2007 Maintenance of genome stability Blazek et al, G&D, 2011 Loss of CycK/Cdk12 causes sensitivity of cells to a variety of DNA damage agents Conclusion I CycK- binds Cdk12 and Cdk13, but not Cdk9 Cdk12 - is a major Ser2 kinase in the CTD of RNAPII -directs expression of a small subset of genes -regulates optimal expression of DNA damage response genes (BRCA1, ATR, FANCI, FANCD2) -is crucial for the maintenance of genome stability -candidate tumor suppressor gene Cdk12 was found among the most often somatically mutated genes in HGSOC Gene No. of Somatic Mutations (%) No. of Pubmed Papers Function P53 302 (96%) 63852 tumor suppressor BRCA1 11 (3%) 9231 tumor suppressor NF1 13 (4%) 3064 tumor suppressor CDK12 9 (3%) 27 ? BRCA2 10 (3%) 5793 tumor suppressor RB1 6 (2%) 2050 tumor suppressor Ovarian cancer - 204 000 new cases worldwide - results in 125 000 deaths per year - relatively low incidence rate, but extremely lethal - highest death-to-incidence ratio among cancers - overall five-year survival probability in about 42% - 70% of deaths are patients with advanced-stage high-grade serous ovarian carcinoma (HGSOC) Vaugham et al., Nat. Rev Cancer, 2011 Year Diagnosed High-grade serous ovarian carcinoma (HGSOC) Vaughan et al, Nature Rev Cancer 2012 - Narrow mutational spectrum - p53 mutated in 96% of patients - recurrent mutations in eight genes including BRCA1/2 ~ 50% of patients have a defect in homologous recombination (HR) DNA repair pathway potentially sensitive to PARP inhibitors therapy - Defect in HR - BRCA1/2 mutations and BRCA1 epigenetic silencing - Fanconi anemia genes mutations (FANCI, FANCD2, FANCA) - Rad family genes mutations - DDR genes mutations (ATR, ATM, Chek1, Chek2) What is the role of CDK12???? HGSOC-related mutations in Cdk12 are clustered in its kinase domain and lead to potential loss of Cdk12 function Carter et al, Nature Biotechnology, 2012 Most of the HGSOC-related Cdk12 mutations are homozygous Adapted from Kohoutek and Blazek, Cell Div., 2012 KD=Kinase Domain (aa 719-1051) W719del E928fs R882L E901C G909R K975E L996F T1014del L122fs Q602del Cdk12 forms a complex with its activating Cyclin, Cyclin K (CycK) Blazek et al, G&D, 2011 Bartkowiak et al, G&D, 2010 Bosken et al, Nature Comm, 2014 Alberts et al, Mol Biol of Cell, 2002 Most of Cdk12 mutations in HGSOC abrogate the kinase activity of Cdk12 and some lead to defective interaction between CycK and Cdk12 Ekumi, Paculova et al, NAR 2015 Structural insights into the detrimental effects of Cdk12 mutations on the kinase activity of Cdk12 Ekumi, Paculova et al, NAR 2015 Cdk12 mutations in HGSOC decrease the transcriptional activation by Cdk12 in reporter assay Ekumi, Paculova et al, NAR 2015 Cdk12 mutations in HGSOC patient samples cause downregulation of genes of the homologous recombination (HR) repair pathway Ekumi, Paculova et al, NAR 2015 Depletion of Cdk12 results in downregulation of HR genes in cell lines Ekumi, Paculova et al, NAR 2015 Cdk12 is recruited to the DDR genes and regulates Ser2 phosphorylation of the CTD of the RNAPII Ekumi, Paculova et al, NAR 2015 Lord and Ashworth, Nature 2012 Double-strand breaks are repaired by HR or by nonhomologous end-joining (NHEJ) Cdk12 lesions disable the frequency of the repair of doublestrand breaks in DNA by HR Ekumi, Paculova et al, NAR 2015 Normal cells HR-deficient cancer cells (mutated HR genes) PARP inhibitors selectively kill HR-deficient cancer cells by inhibiting alternative NHEJ pathway DeathSurvival PARPi PARPi Adapted from Ashworth, JCI, 2008 Normal cells HR-deficient cancer cells (mutated HR genes) Depletion of Cdk12 sensitizes ovarian cancer cells to PARP inhibitors Individual Mutations in HR-related Genes: BRCA1/2 FANCI ATM ATR MDC1 Chek1 Rad51D …. Defect in HR HGSOC (PARPi responsive) Individual Mutations in: Cdk12 Common Down-regulation of HR-related Genes: FANCI ATM ATR MDC1 Chek1 Rad51D (BRCA1) Defect in HR HGSOC (PARPi responsive) Cdk12 mutations cause a defect in HR pathway by collective down-regulation of critical HR genes Farmer et al, Nature, 2005 McCabe et al, Cancer Research, 2006 Morrison et al, EMBO J, 2007 The Cancer Genome Atlas, Nature, 2011 Lord and Ashworth, Nature, 2012 Blazek et al, G&D, 2011 Blazek, Cell Cycle, 2012 Bajrami et al, Cancer Research, 2014 Joshi et al, JBC, 2014 Ekumi, Paculova et al, NAR, 2015 Conclusions II - Most HGSOC Cdk12 mutations interfere with Cdk12/CycK complex formation - Mutations likely cause structural rearrangements detrimental to Cdk12 activation - Patient samples containing the Cdk12 mutations have diminished expression of HR genes (ATM, ATR, Rad51D, FANCI) - Cells with Cdk12 mutations fail to repair DNA double-strand breaks via HR - Cdk12 mutations have a potential to be markers of PARP inhibitor therapy in patients with HGSOC Acknowledgements CEITEC/Masaryk University Brno Koen Bartholomeeusen Pavla Gajduskova Milan Hluchy Hana Paculova Kveta Pilarova Jana Rybarikova Dalibor Blazek Funding: Marsha Rivkin Center For Ovarian Cancer Research Czech Science Foundation (GACR) SoMoPro CEITEC/Masaryk University University of Helsinki Kingsley Ekumi Tina Lenasi Matjaz Barboric Caesar Bonn Christian Bosken Matthias Geyer Masaryk University Brno Vendula Pospichalova Vita Bryja