Molecular Cell Biology (Bio 5068) Cell Cycle I Ron Bose, MD PhD November 10, 2016
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Molecular Cell Biology (Bio 5068) Cell Cycle I
Ron Bose, MD PhD November 10, 2016
G1
M
G2
S
CELL DIVISION CYCLE
DISCOVERY AND NAMING OF CYCLINS
A protein (called “cyclin”) was observed to increase as cells approached mitosis, peak in mitosis and then precipitously disappear as cells exited mitosis.
Two proteins (cyclins A and B) increased as cells approached mitosis, peaked in mitosis and precipitously disappeared as cells exited mitosis.
The cell cycle is primarily regulated by cyclically activated protein kinases
Figure 17-15, 17-16 Molecular Biology of the Cell, 4th Edition
Presenter
Presentation Notes
Figure 17-15 Two key components of the cell-cycle control system A complex of cyclin with Cdk acts as a protein kinase to trigger specific cell-cycle events. Without cyclin, Cdk is inactive. Figure 17-16 A simplified view of the core of the cell-cycle control system Cdk associates successively with different cyclins to trigger the different events of the cycle. Cdk activity is usually terminated by cyclin degradation. For simplicity, only the cyclins that act in S phase (S-cyclin) and M phase (M-cyclin) are shown, and they interact with a single Cdk; as indicated, the resulting cyclin-Cdk complexes are referred to as S-Cdk and M-Cdk, respectively.
Table 17-1. Molecular Biology of the Cell, 4th Edition
Overview of major cyclins and Cdks of vertebrates and yeast
Malumbres M, Nature Reviews Cancer 2009
Evolution of cell cycle control: from yeast to humans
Presenter
Presentation Notes
The Saccharomyces cerevisiae genome encodes about 23 cyclins, which regulate six proline-directed serine/threonine protein kinases: Cdc28 (cell division control 28), Pho85, Kin28, Srb10 (also known as cyclin-dependent kinase 8 (Cdk8) and Ccn3), Sgv1 (also known as Bur1) and Ctk1. Two of these kinases, Cdc28 and Pho85, are activated by large cyclin families, whereas the other kinases associate with a single dedicated cyclin. Cdc28 and its associated cyclins are essential for driving the cell cycle. The multifunctional kinase Pho85 regulates G1 progression, cell polarity and the actin cytoskeleton, transcription, phosphate and glycogen metabolism, and senses changes in the environment115. The four yeast CDKs that associate with a single cyclin have close ties with the transcriptional machinery and probably control gene expression through regulation of the RNA polymerase II holoenzyme and transcription factors. In humans, the CDK family is composed of 13 members (Box 2) that interact with at least 29 cyclins or cyclin-related proteins5. An additional family of five proteins (known as Ringo or Speedy) with structural but not sequence homology to cyclins, have been found in vertebrates but not in S. cerevisiae, Caenorhabditis elegans or Drosophila melanogaster116. Whereas some transcriptional CDKs such as CDK7, CDK8 and CDK9, have not diverged much from their yeast orthologues, cell cycle CDKs have evolved in metazoans to generate new CDK subfamilies, such as CDK4 and CDK6 (see the figure). In S. cerevisiae, nine different cyclins (Cln and Clb subgroups) bind and activate Cdc28. Progression through G1 requires Cdc28 and at least one of the G1 cyclins (Cln1, Cln2 and Cln3), whereas efficient DNA replication requires Cdc28 and the early expressed B-type cyclins (Clb5 and Clb6). Functional complexes of Cdc28 and mitotic cyclins (Clb1, Clb2, Clb3 and Clb4) are essential for mitotic events117. Pho85, on the other hand, associates with 10 cyclins (Pcl subgroup) that function in nutrient sensing. At least four of these Pcls (Pcl1, Pcl2, Pcl7 and Pcl9) exhibit cell cycle periodic expression from early G1 to S phase. A specific function for Pho85 in sensing mitogenic signals for G1 progression has been suggested115. The increased number of cyclins in the mammalian genome has resulted in a large variety of CDK–cyclin complexes5, 116. However, only ten cyclins (three D-type, two E-type, two A-type and 3 B-type cyclins) are known to be directly involved in driving the mammalian cell cycle.
Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins
Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition
Why is cell cycle progression governed primarily by inhibitory regulation?
Presenter
Presentation Notes
Figure 17-18 The regulation of Cdk activity by inhibitory phosphorylation The active cyclin-Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 results in activation of the cyclin-Cdk complex. For simplicity, only one inhibitory phosphate is shown. The activating phosphate is added by CAK, as shown in Figure 17-17. Figure 17-19 The inhibition of a cyclin-Cdk complex by a CKI This drawing is based on the three-dimensional structure of the human cyclin A-Cdk2 complex bound to the CKI p27, as determined by x-ray crystallography. The p27 binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk. It also inserts into the ATP-binding site, further inhibiting the enzyme activity.
Figure 17-20. Molecular Biology of the Cell, 4th Edition
Cell cycle control depends on cyclical proteolysis
Presenter
Presentation Notes
Figure 17-20 The control of proteolysis by SCF and APC during the cell cycle (A) The phosphorylation of a target protein, such as the CKI shown, allows the protein to be recognized by SCF, which is constitutively active. With the help of two additional proteins called E1 and E2, SCF serves as a ubiquitin ligase that transfers multiple ubiquitin molecules onto the CKI protein. The ubiquitylated CKI protein is then immediately recognized and degraded in a proteasome. (B) M-cyclin ubiquitylation is performed by APC, which is activated in late mitosis by the addition of an activating subunit to the complex. Both SCF and APC contain binding sites that recognize specific amino acid sequences of the target protein.
UBIQUITIN-MEDIATED PROTEOLYIS
E1 (Ubiquitin activating enzyme) Binds to Ubiquitin in an ATP-dependent manner Passes Ubiquitin to E2 E2(Ubiquitin conjugating enzyme or UBC) At least 12 in yeast some are specific to a given target E3 (ubiquitin protein ligase) Large complex in both clam (cyclosome) and in frog (APC = anaphase promoting complex). Final transfer of ubiquitin to substrate can be mediated by E2 alone or E2 acting in concert with E3 Proteosome (26S complex) Structure from archaebacterium solved.
Deshaies RJ and Joazeiro CA. Annu Rev Biochem. 2009
UBIQUITIN-MEDIATED PROTEOLYSIS
APC = A naphase P romoting C omplex Required for degradation of substrates at Metaphase to Anaphase transition ( ie : B-type Cyclins and securin)
Cdc20 : targets cyclin A and B-type Cyclins, securin Cdh1 / Hct1 : targets Plk1 and B-type cyclins
substrate
Ring finger UBC ( E2 )
for ubiquitination
Apc10
Cdc20 Apcx
Apc4
Apc5
Apc7
Cdc27 Apc3
Apc1 / BimE
Cdc23 Apc8
Cdc16 Apc6
Have D or KEN Box
Cullin = Apc2
Apc11
or Cdh1 / Hct1
WD repeat- containing proteins
Ubiq
Ubiq Ubiq
Ubiq
Components: F Box: adapter Brings substrate to E3 ligase. F-box binds to Skp1 Additional protein interaction domains (PID: WD repeat, leucine-rich repeat) binds to substrate E2: UBIQ. Conjugating enzyme (transfers UB to substrate) Skp1: Bridges F-box to cullin Cullin: Organizes and activates E3 complex Recruits E2-UBIQ conjugating enzyme Ring finger protein Participates in E2 binding and catalysis
SCF Ubiquitin Ligases
O'Connell BC, Harper JW. Curr Opin Cell Biol. 2007
SCF E3 Ubiquitin Ligases
CELL CYCLE REGULATION OF Cdc2
Phosphatase(s)
P T161
P P P
T14 Y15 T161
Cyclin B
Cdc2
Inhibitory kinase(s)
Activating Kinase(s)
Cyclin B
Cdc2
Cyclin B
Cdc2 Cdc2
Cyclin B
Cdc2
INACTIVE INACTIVE INACTIVE
INACTIVE ACTIVE
Protein-protein interactions
Reversible phosphorylation
Ubiquitin-mediated proteolyis
Cyclin-dependent Kinase Inhibitor Proteins (CKI’s) 1. CIP/KIP family (p21Cip1, p27Kip1, p57Kip2):
a. Binds to Cdk2 and inhibits activity. b. Binds Cdk4/6 and helps assemble complexes with
cyclins. 2. INK4 family (p16, p15, p18, p19).
a. Specific for Cdk4 and Cdk6. b. Binds Cdk subunit alone and prevents cyclin binding c. Bind and inhibit Cdk4/6-Cyclin D heterodimers.
G1 Control
Cdk 4 & 6 Cyclin D1, 2, 3
Cdk2 Cyclin E
G1
M
G2
S
Cip/Kip proteins (p21, p27, p57)
INK4a proteins (p15,16, 18, 19)
Assembly & Sequestration
Mechanisms controlling G1/S-phase transition
Figure 17-30. Molecular Biology of the Cell, 4th Edition
MITOGENIC or
HORMONAL SIGNALS
CYCLIN D Stability and CYCLIN D-dependent Kinases (Cdk4/Cdk6)
Rb1 E2F
Transcription factor
Presenter
Presentation Notes
G1-Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation. This inactivates Rb, freeing E2F to activate the transcription of S-phase genes, including the genes for a G1/S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G1/S-Cdk and S-Cdk activities further enhances Rb phosphorylation, forming a positive feedback loop. E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop.
MITOGENIC SIGNALS
CYCLIN D STABILITY CYCLIN D-DEPENDENT KINASES (Cdk4/Cdk6)
RB E2F
E2F
CYCLIN E
CYCLIN E CDK2
CYCLIN A & S-PHASE GENES
S-Phase S-PHASE GENES
Relief of Rb- mediated transcriptional repression
S-Phase
G1 Control
MITOGENIC SIGNALS
CYCLIN D STABILITY CYCLIN D-DEPENDENT KINASES (Cdk4/Cdk6)
RB E2F
E2F
CYCLIN E
CYCLIN E CDK2
CYCLIN A & S-PHASE GENES
p27KIP1 p27KIP1-Phosphorylation Ubiq-Mediated proteolysis
Assembly & Sequestration
S-Phase S-PHASE GENES
Relief of Rb- mediated transcriptional repression
S-Phase
G1 Control
Checkpoints What are they? How were they defined? How does their derailment contribute to cancer?
CHECKPOINTS
G1 G1
M M
G2 G2
S S
DNA DAMAGE
DNA DAMAGE UNREPLICATED DNA
IMPROPER SPINDLE ASSEMBLY
STOP!
Checkpoints: intracellular signaling pathways that determine if previous steps are complete before proceeding onto the next stage (complete DNA synthesis
before entering mitosis; spindles must be assembled before exiting metaphase and entering into anaphase) and whether there has been any damage to the DNA.
DNA damage checkpoint: integrity of DNA DNA damage is repaired before entering S, completing S or entering M.
DNA replication checkpoint: replication state of DNA Complete DNA synthesis before mitosis.
Spindle assembly checkpoint: integrity of spindle spindles must be assembled before exiting metaphase into anaphase.
G1
M
G2
S
G1-PHASE CHECKPOINT
S-PHASE CHECKPOINT
G2-PHASE CHECKPOINT
DNA DAMAGE RESPONSE PATHWAY
STOP!
CELLULAR RESPONSES TO CHECKPOINT ACTIVATION (IR, etoposide, HU, gemcitibine, irinotecan, carboplatin…)
G1 S G2 M
CHECKPOINTS
APOPTOSIS
SENESCENCE
TEMPORARY CELL CYCLE ARREST
& activation of DNA repair pathways
IR Etoposide Gemcitabine Cytarabine 5-Fluorouracil
Irinotecan Topotecan
Cisplatin Carboplatin
ATM ATR
CHK2 p53 CHK1
RAD51 FAND2 FANCE
p21 14-3-3σ
BAX PUMA
CDC25A
Apoptosis Cell Cycle Arrest DNA Repair
Signaling Cascade
Cellular Response
Chemo- & Radio-therapy
Signal
Fig. 2
Senescence
DNA DAMAGE CHECKPOINTS
Chk1
Cyclin E / Cdk2
G1 S G2 G1-checkpoint S-phase checkpoint G2 checkpoint
M DEATH
Cdc25A
DNA DSBs ssDNA
ATM
Mdm2 p53
p21, 14-3-3σ
IR/VP16 replication stress
ATR
Chk2
Cyclin B/Cdk1
Overproduced in certain cancers. Inactivated in certain cancers.
DNA damage leads to cell cycle arrest in G1
Figure 17-33. Molecular Biology of the Cell, 4th Edition
Presenter
Presentation Notes
When DNA is damaged, protein kinases that phosphorylate p53 are activated. Mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. Phosphorylation of p53 blocks its binding to Mdm2; as a result, p53 accumulates to high levels and stimulates transcription of the gene that encodes the CKI protein p21. The p21 binds and inactivates G1/S-Cdk and S-Cdk complexes, arresting the cell in G1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes an increase in p53 (not shown).
Figure 17-41. Molecular Biology of the Cell, 4th Edition
Mitogens stimulate cell division
Presenter
Presentation Notes
The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a gene encoding a subunit of the SCF ubiquitin ligase. The resulting increase in G1-Cdk and G1/S-Cdk activities promotes Rb phosphorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry (see Figure 17-30). Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. Although, for simplicity, Myc is shown as a monomer, it functions as a heterodimer with another protein called Max.
Figure 17-42. Molecular Biology of the Cell, 4th Edition
Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death
Presenter
Presentation Notes
Figure 17-42 Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic pathways Abnormally high levels of Myc cause the activation of p19ARF, which binds and inhibits Mdm2 and thereby causes increased p53 levels (see Figure 17-33). Depending on the cell type and extracellular conditions, p53 then causes either cell-cycle arrest or apoptosis.
Figure 17-47. Molecular Biology of the Cell, 4th Edition
Extracellular Survival Factors Suppress Apoptosis
Presenter
Presentation Notes
(A) In mammalian cells, the binding of some survival factors to cell-surface receptors leads to the activation of various protein kinases, including protein kinase B (PKB), that phosphorylate and inactivate the Bcl-2 family member Bad. When not phosphorylated, Bad promotes apoptosis by binding and inhibiting Bcl-2. Once phosphorylated, Bad dissociates, freeing Bcl-2 to suppress apoptosis. As indicated, PKB also suppresses death by phosphorylating and thereby inhibiting gene regulatory proteins of the Forkhead family that stimulate the transcription of genes that encode proteins that promote apoptosis.
Malumbres M, Nature Reviews Cancer 2001
Cell cycle regulators are frequently disrupted in cancer
O’Leary et al., Nature Reviews Clinical Oncology 2016
Overview of CDK inhibitors in clinical development for cancer therapy
Turner et al, NEJM 2015
NEJM Nov. 3, 2016
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