Cell Cycle Regulation + + +

Continuous Duplication Cytoplasmic Cycle Cell Growth Cytokinesis 2 1 Quantum Duplication Chromosome Cycle DNA Replication Mitosis 2 1

The Basic Problem S phase G1 G2 Mitosis

Cell Cycle Stages G1(G0) - S - G2 - M How do we determine which stage of the cycle a cell is in? 1. FACS analysis Fluorescence Activated Cell Sorting 2. Incorporation of radioactive or epitope tagged nucleotides (BrdU) 3. Landmarks Nuclear envelope breakdown Condensed chromosomes Spindle elongation Determining G0 vs G1 can be difficult.

G1 4 Cells sense their environment - nutritional - geometrical (cell-cell contact) - physical (size am I big enough) - regulatory (growth factors) If all is well, the cell will commit to a new cell cycle and pass START in yeast or the R-point (restriction) in mammals. The later stages of G1 involve preparation for S phase and mitosis. These include induction of gene expression and duplication of centrosomes (MTOC).

S phase 5 Cells initiate DNA synthesis. They fire early origins early and late origins late. Early Late Cell growth continues. During S phase cells must have a mechanism to prevent activation of mitosis until DNA replication is completed. In addition, chromosomes must be replicated once and only once per S phase. Origins fire only once except in special cases.

G2 6 No major cytological events Probably a sensing period for accumulation of information leading to the commitment to Mitosis. Proteins needed for Mitosis are synthesized in G2.

Mitosis Seven parts. Each of these is carefully regulated Prophase Prometaphase Metaphase Anaphase A Anaphase B Telophase Cytokinesis Each of these is carefully regulated so as to occur in the proper order.

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

Interphase Early Prophase Late Prophase Prometaphase Metaphase Anaphase A Anaphase B Telophase

Cell Cycle Transitions State A State B

Cell Cycle Transitions State A State B

Mutual Incompatibility Cell Cycle Transitions State A State B Metastable States Mutual Incompatibility

Cell Cycle Transitions State A State B State C Inhibitory Barriers

Cell Cycle Transitions State A State B

Cell Cycle Transitions Cdc Mutants X State A State B

X X Cell Cycle Transitions State A State B A Checkpoint Pathway Creates a Dependency Relationship

Cell Cycle Transitions State A State B Self-Reinforcement

Cell Cycle Transitions State A State B Self-Reinforcement

Cell Cycle Transitions Mitosis G1 S G2 Meta Ana Telo

Cell Cycle Transitions G1 S G2 Meta Ana Telo Cdk1 SCF Cdk1 SCF Esp1 APC APC

Cell Cycle Transitions G1 S G2 Meta Ana Telo Cdk Cdk Esp1 APC/C CKI Wee1 Pds1 Cdk1

Cell Cycle Genetics cdc mutant 24°C 37°C YEAST The genetic system. Advantages 1. Eukaryotic cell cycle 2. Haploid---Diploid 3. Transformable 4. Reverse genetics CDC Mutants 1. Conditional lethal mutants cdc mutant 2. Arrest the cell cycle at a unique position. Can be used for four purposes 1) Make a molecular map of the order of 24°C function of cdc protein and landmark events. 2) Make a determination of whether certain processes are dependent or not. 3) Identify genes important for a particular 37°C process. 4) Identify other genes in the process by reversion analysis.

Cdk1 = cdc2 + Cdc28 cdc2 was identified in S. pombe as a mutant that arrests in G2 and gives rise to long cells. A critical experiment identified a dominant allele of cdc2 that cause the cell cycle to accelerate rather than stop, yielding shorter cells. Why is this so important? CDC28 was identified in S. cerevisiae -protein kinase Two stop points G1 and G2 (the original allele had only one arrest point in G1) How does one protein regulate two completely different processes?

The Relationship Between Cyclins and MPF mitosis interphase mitosis interphase Cyclin A Cyclin B RNR

Cyclins Cyclins are periodically accumulated during the cycle, then rapidly destroyed. I M I M I M 1 2 3 MPF MPF activity peaked with cyclin levels. Cyclins are a regulatory component of MPF.

Cyclins are the regulatory subunit of cyclin-dependent kinases (Cdk) Cyclins bind to Cdks and activate the kinase and, in some circumstances, control their substrate specificity Activation of Cdks controls certain cell cycle transitions G1 S phase Mitotic Cyclin/Cdk Cyclins/Cdk Cyclins/Cdk Mitotic G1 S G2 M Exit G1 Cdk S Cdk M Cdk All Cdks ON ON ON OFF

S. cerevisiae Cyclin/Cdk Activity CLN1 CLB5 CLB3 CLB1 CLN3 CLN2 CLB6 CLB4 CLB2 2 Classes of Cyclins G1 Cyclins CLN Cln1, 2, 3 S phase CLB Clb5, 6 Mitotic CLB Clb, 1, 2, 3, 4, G1 S G2 M Redundancy among cyclins Deletion of one Cln or Clb has little effect D cln1, 2, 3, ------ G1 Arrest D clb1, 2, 3, 4 ------ G2 Arrest Cyclins gain functions later in the cycle. Clbs can carry out the function of Clns in some circumstances Clb1-4 can carryout the functions of Clb5,6 but Clb5,6 cannot function in place of Clb1-4.

The Start of START What exactly is START? Cell size SBF ? ? Sic1 ? ? Auto-activation Loop Cell size Cdc28 Cln1/2 SBF ? ? (Swi4/6) Cdc28 Cln3 Sic1 ? ? Cdc28 Clb5/6 MBF S phase Nutrients uORF CLN3 Auto-activation Loop What exactly is START?

The end of START and the start of S phase Budding + Centrosome Duplication Sic1 is made during the previous mitosis SCF Sic1 SBF Cdc28 Cln1/2 Cdc28 Clb5/6 Clb1-4 MBF Cdc28 Nutrients G1 S G2 M Sic1 ensures that S is dependent upon G1 cyclins

Ubiquitin Conjugation Cascade Ub Activating Enzyme E2 S Ub Ub Conjugating Enzyme E3 Ub Ligase Ub n substrate Ub n substrate Destruction by the 26S Proteasome

SCFCdc4 Sic1 degradation through the SCFCdc4 pathway Sic1 Sic1 Sic1 Rbx1 Signal C dc 34 E2 P Cln/Cdc28 C dc 53 Sic1 Sic1 P S kp 1 Clb5 Cdc28 Clb5 Cdc28 + Cdc4 SCFCdc4 E1-S-Ub Ub P Rbx1 N S-Ub P Sic1 C dc 34 C dc 53 Ub P Ub N S kp 1 Sic1 Proteasome P Clb5 Cdc28 Clb5 Cdc28 S phase Cdc4

The F-box Hypothesis Rbx1 Cdc34 Cdc53 Substrate Function F Sic1 Cdk Inhibitor Cdc4 S S kp kp 1 1 F Cln2 Cyclin Grr1 F Unknown Targets Other F-box Proteins

Cell Cycle Control Tumorigenesis F-box Proteins Signaling/ Development G1 cyclins (Cln1) Bud site selection p27 Cdk Inhibitors (Sic1) Cyclin E DNA replication (Cdc6) Grr1 Skp2 Fbw7 Plant flowering Cdc4 UFO Circadian rhythms in plants Auxin response in plants F-box Proteins FKF1 TIR1 Sel-10 Met30 Cell Fate (Notch) Aminoacid biosynthesis (Met4) Dactylin b-TRCP NFkB Activation (IkB) Limb development Wnt Pathway (b-catenin) Hedgehog (Ci) Development Signaling/ Transcription

RING-Finger Based Ubiquitin Ligases Rbx1 Apc11 Ub Cdc34 Ub E2 Apc2 Cdc53/ Cul1 E2 Ub Ub n Ub n substrate Skp1 substrate P P RING F SCF, VCB Anaphase Promoting Complex Simple RING-E3s F-box Proteins BC-box Proteins 5 different Cullins MDM2 Cbl BRCA1 Parkin Cdc20 Cdh1

Two Major Classes of E3s HECT Family RING Superfamily SCFs APC Simple E6-AP (Angelman’s Syndrome) Smurf1 (Smad destruction) Itch (Notch destruction) Rsp5 (membrane protein endocytosis) SCFs APC Simple RING E3s

Making the cycle go forward: Cell Cycle Logic Making the cycle go forward: SCF Grr1 SBF Cdc28 Cln1/2 SCF Cdc4 (Swi4/6) Sic1 SCF Cdc4 Cdc28 Clb5/6 S phase MBF Time

Activation of Mitotic Cyclin-Dependent Kinases Mitotic Entry Activation of Mitotic Cyclin-Dependent Kinases

Cdk Regulation * * cyclin Cdk Cks CKIs Cdk Inhibitors Synthesis Destruction SCF Complexes Phosphatase Kap1 Kinases cyclin CAK T161 Civ Synthesis Destruction Cdk Cks SCF Complexes T14 Y15 CKIs Cdk Inhibitors Kinases Phosphatases Sic1 Wee1 * Cdc25 * Far1 Mik1 Pyp3 Rum1 Myt1 p21, p27, p57 p16, p15, p18, p19

- G2 M + Mitotic Entry in S. pombe and Mammals Phosphorylation Regulation of Cdc2 during Mitosis (Cdc13) cyclin B Cdc2 (Cdk1) cyclin B/Cdc2 CAK + cyclin B/Cdc2 (T160-P) ACTIVE KINASE - Tyrosine Wee1 Kinases Mik1 cyclin B/Cdc2 Y-P (Inactive Y15-P) + Phosphotyrosine Cdc25 Phosphatase cyclin B/Cdc2 ACTIVE KINASE G2 M

- - + G2 M + Cell Cycle Logic Autoactivation of Cdc2 makes mitosis irreversible (Cdc13) cyclin B Cdc2 (Cdk1) cyclin B/Cdc2 CAK + cyclin B/Cdc2 (T160-P) - Tyrosine Wee1 Kinases Mik1 cyclin B/Cdc2 Y-P (Inactive Y15-P) - + Phosphotyrosine Cdc25 Phosphatase + cyclin B/Cdc2 (active) G2 M

APC/Cyclosome The APC is a complex ubiquitin ligase that is required for anaphase entry and mitotic exit. Like the SCF, it has substrate specificity components called Cdc20 and Cdh1/Hct1, 2 WD40 repeat proteins. The regulation of these specificity components is critical.

Anaphase Entry and Exit APC APC Clb5 Chromosomes Cdc20 Clb2* Cdh1 OK ? Pds1 Cohesion Clbs Factors Cdk1 Mitotic Exit Anaphase

Chromosome Cohesion APC Pds1 Pds1 Esp1 Esp1 Destruction by the Ub Ub Ub Destruction by the Cdc20 Pds1 APC 26S Proteosome Pds1 has a destruction box which allows Pds1 it to be recognized by the APC Securin Esp1 Separin Esp1 Cohesin Separin Anaphase

Cohesion in Mammals Securin = Pds1 Separin = Esp1 Cohesin = Scc1 + APC Separin (Esp1)

Mitotic Exit Mitosis Mitotic Exit Cytokinesis & G1 Entry After anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks. Mitosis Mitotic Exit Cytokinesis & G1 Entry High Low Cyclin B/Cdk Activity Cyclin B/Cdk Activity Low High Cdc14 Phosphatase Cdc14 Phosphatase

Mitotic Exit After anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks. This involves activation of the Cdh1 form of the APC. Cdh1 Cdh1 P Cdh1 Active Clb Cdc14 Cdk1 (Phosphatase) Inactive Cdh1 APC MEN Cdc14 Cdc14 Inactive Active Clb/Cdk1 Cdc14 Swi5 Swi5 P Swi5 Sic1 Transcription Factor Clb Cytoplasmic Nuclear Cdk1 Inactive Active Mitotic Exit

Cdc14 Activation for Mitotic Exit The activation of Cdc14 is the key event in execution of mitotic exit. During S, G2 and Pre-anaphase, Cdc14 is held tethered in an inactive complex in the nucleolus. When Anaphase is executed, Cdc14 is released and goes throughout the nucleus and cytoplasm to dephosphorylate key Cdk1 substrates. Nucleolar Spindle Cdc14 MEN Cfi1 Cfi1 Cdc14 Cdc14 (Net1) (Net1) Inactive Active The mitotic exit network (MEN) consists of several protein kinases and a G-protein Tem1. How it MEN regulated is not known.

How Mitotic Exit is Coupled to Anaphase Spindle Pole Body Tem1-GDP Lte1 (GEF) Bfa1/Bub2 (GAP) Tem1-GTP Mitotic Exit Network Cdc15 Dbf2, Mob1 Clb2 Cdc14 (Nucleolus) Cdc14 (Released) Mitotic Exit Sic1

The Tem1-bearing SPB migrates into the daughter cell to encounter Lte1 Lte1 in Red Tem1 in Red Spindle in Green

Mitotic Exit Summary 1. When anaphase occurs, Tem1 on the SPB is thrust into the daughter cell where it encounters the GEF, Lte1, Tem1 is converted to the active GTP form. 2. Active Tem1 activates Cdc15 and MEN, which causes the release of the Cdc14 phosphatase from the nucleolus where it is inhibited. 3. Cdc14 dephosphorylates Cdh1 to activate the APC to destroy Clbs, it also activates the synthesis of Sic1, a Cdk inhibitor. 4. Together, the APC and SIC1 turn off Cdk activity to initiate mitotic exit. Cell move from high CDK, low CDC14 state to a Low CDK, high Cdc14 state. To re-enter the next cell cycle they need to turn off Cdc14 to re-establish the null state, CDK off, Cdc14 off, APC off, making cells permissive for Clb activation of S phase.

How does the cycle move forward? - Positive amplification loops - Feedback inhibition 1) Clns activate their own transcription 2) Once Clns provide sufficient activity to pass START, they activate a ubiquitin proteolysis pathway that destroys an inhibitor of Clb kinase activity, Sic1. 3) Clb/Cdc28 kinase activate Clb transcription and repress Cln transcription. 4) Clb/kinase activate S phase. 5) Once S phase is complete, Clb kinases activate mitosis. 6) Once chromosomes are properly aligned at the metaphase plate, a ubiquitin proteolysis pathway is activated that destroys Clbs but not Clns and resets the cycle. 7)Clb destruction allows PRC complexes to form. 8) Cln kinase activity is required to shut off the Clb proteolysis pathway to allow S entry in the next cell cycle. This allows Pds1 to be synthesized again which recruits Esp1 into the nucleus. In Mammals Cyclin B/Cdc2 can help activate itself by turning on an activating phosphatase and turning off an inhibitory kinase

The Rao and Johnson Cell Fusion Experiments Cell Cycle Regulation M cells + G1, S, or G2 cells M - Mitotic state is dominant. S cells + G1 G1 cells enter S S cells + G2 G2 cells do not enter S, but do not enter mitosis until the S-phase nucleus has entered G2. - Block to re-replication - Inhibitor of mitosis produced by S phase cells G1 cells + G2 Like S above. - G1 cells also block mitosis

Cell Cycle Checkpoints Definition: "A checkpoint is a biochemical pathway that ensures dependence of one process on another process that is otherwise biochemically unrelated." B C Intrinsic A Mechanism D E Extrinsic Mechanism Damage

Why are checkpoints important? Checkpoints control the order and timing of events. In some cases the natural timing of events can allow the proper order of events in the absence of a checkpoint. However, the fidelity is often compromised. The accumulation of errors, whether due to entering DNA replication in the presence of damage, or mis- segregating a chromosome is deleterious to the reproductive fitness of unicellular organisms, and in multicellular organisms may lead to uncontrolled cell proliferation and cancer.

Checkpoints in S. c. DNA Damage Checkpoints Spindle Assembly Checkpoints S phase Checkpoints Size Checkpoints G1/M Checkpoint Morphology Checkpoint Meiotic Checkpoints Checkpoints are defined by loss of function mutations that relieve the dependency of two events. cdc13 ts mutants cdc13 rad9 mutants

The Spindle Assembly Checkpoint The proper assembly of a spindle is sensed by a group of proteins called Mad or Bub located on the kinetochore. These proteins send a signal to inhibit the APC. Mutant Hunt - benomyl sensitive mutants that continue to cycle in the presence of benomyl. ben WT ben mad or bub mutants Metaphase Anaphase A/B Misaligned Chromosomes Scc1 Mps1 Mad1,2,3 Bub1,2,3 Esp1 Cdc20 APC Pds1

The Spindle Assembly Checkpoint What is being sensed? Kinetochore - Microtubule Attachment Tension and bipolar attachment When tension is not present at sister chromatids, a Mad/Bub- dependent phosphorylation occurs on the kinetochore. This is thought to be part of the signal used to turn off the APC.

Signal Transduction Signal Sensor Transducer Effector

DNA Damage Response Pathways SIGNALS Conserved Families SENSORS PCNA- & RFC-like Proteins Mediators (BRCT proteins, Mrc1/Claspin) TRANSDUCERS Kinases: PIK ATM + ATR EFFECTORS PK CHK1 and CHK2 STOP Cell Cycle Arrest Transcription Apoptosis DNA Repair

The DNA Damage Response in Humans Hus1 Rad17 P RFC P Rad1 ATRIP ATR Rad9 PC Claspin P P P P BRCA1 BLM NBS1 Repair Proteins Chk1 Chk2 P P p53 P P Cdc25 p21 DNA replication proteins? Cdc2/Cyclin B G1 S G2 M

DNA Damage Checkpoints - Sensing Damage ATRIP ATR Rad17 RFC ATP P P ATRIP Hus1 Hus1 ATR Rad17 Rad1 Rad1 RFC Rad9 Rad9

Checkpoint Responses ATR and RC-PC Engagement Activates Checkpoint Hus1 Hus1 P ATRIP Rad17 Rad1 Rad1 RFC ATR Rad9 Rad9 P P P P P P Mediators Chk1 Chk2 BRCA1 Nbs1 Checkpoint Responses

G1 Arrest in Mammals Cdk activity is rate limiting for S phase entry and is the target for checkpoint control. DNA Damage Chk1,2 ATM/ATR Cdc25A ? Chk2 G1 Cyclin Mdm2 p53 p53* p21 Cdks or Apoptosis p53 levels increase in response to DNA damage and activate transcription of p21

How is p53 activated? - Relief of repression. MDM2 binds p53 and targets it for ubiquitin-mediated proteolysis. p53 transcriptionally regulates MDM2 to make a feedback loop. Mdm2 RING Finger Ubiquitin Ligase p53 Mdm2 transcription In response to DNA damage, both p53 and Mdm2 are phosphorylated, causing a disruption in Mdm2 binding, thereby allowing p53 to both increase in abundance and become transcriptionally active. During activation, p53 increases the amount of Mdm2 protein to return to low p53 levels when the signal is eventually turned off. This also explains why p53 levels are so high in tumors in which p53 is mutant, no Mdm2 is made.

Mitosis S/G2 G2 Arrest in Mammals Cdc25 is regulated by Chk1 phosphorylation DNA Damage Active ATR Inactive Chk1 Ser216 Ser216 14-3-3 P Cdc25C Cdc25C ON OFF Nuclear Cytoplasmic Mitosis S/G2

G2 Arrest in S. pombe + Mammals Cdk activity is rate limiting for entry into Mitosis and is the target for checkpoint control. DNA Damage 14-3-3 Cytoplasm OFF Cdc25 ATM or ATR P Chk1 Chk1* Nucleus ON Chk2 Chk2* Cdc25 Wee1 OFF ON cyclin B cyclin B Cdc2 Y-P Cdc2 OFF ON p53* p21 Mitosis

* * Mechanism of pre-anaphase arrest in response to DNA damage Mitotic S. pombe Mammals S. cerevisiae rad3 rad26 ATR MEC1 DDC2 crb2 Mediator RAD9 Chk Kinases CHK1 RAD53 chk1 * Effectors cdc25 * PDS1 This the genetic DNA damage pathway in budding yeast, Pds1 pathway has been shown is parallel to Rad53 pathway, but there is cross talk between them, Not only Pds1 will block the anaphase entry in response to DNA damage, recent data suggests it also inhibit the mitotic exit. Therefore the regulation of Pds1 in response to DNA damage is crucial to achieve the cell cycle arrest. Now, I will tell you the important finding in our lab that we find the kinase Chk1 that might directly regulate Pds1 by phosphorylation in response to DNA damage. This work was done mainly by two postdoctors in our lab. CDK cdc2 ESP1 Mitotic Entry Anaphase Entry Mitotic Exit Chk1 phosphorylation of Pds1 protects it from degradation by the APCCdc20

Overall Organization of the Cell Cycle Replication Checkpoint ? G1 S G2 Meta AnaA AnaB Tele S Cdk M Cdk M Cdk Sic1 Pds1 B/Cdk1 SCF APC APC Spindle Cdc20 Cdc14 Cdh1 G1 Cdk Checkpoint Mitosis Cdc20 Cdh1 APC APC SCF APC APC APC ON ON OFF ON ON B/Cdk1 B/Cdk1 B/Cdk1 OFF OFF ON

General Points Cells need to do only a few things absolutely right 1. They must duplicate their chromosomes precisely, i.e. completely but only once per cycle. 2. They must segregate their chromosomes precisely. 3. They must divide their cell in two. General Properties of Cell Cycle Transitions 1. Amplification mechanisms. 2. Out with the old, in with the new. 3. Overcoming inhibitory barriers- Checkpoints. Checkpoints allow the coordination of events.