Cell Cycle. Trends in Cell Biology

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1 Cell Cycle Trends in Cell Biology

2 Cyclic proteolysis Two distinct enzyme complexes proteolysis of cyclin-cdk complexes SCF (Skp1-Cul1-F box) Ubiquitylation and destruction of G1/S-cyclins and CKI proteins that control S-phase initiation Activity is constant during the cell cycle Activity depends on changes in the phosphorylation state of the target protein Only specifically phosphorylated proteins are recognized, ubiquitylated and destroyed

3 Control of Proteolysis by SCF

4 APC (anaphase-promoting complex) Ubiquitylation and proteolysis of M-cyclins and Regulators of mitosis Activity changes at different stages of cell cycle Activity depends on addition of activating subunits to the complex

5 Control of Proteolysis by APC

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7 Families of Cyclins and Cyclin-Dependent Kinases Progression through the G1 restriction point is controlled by complexes of Cdk4 and Cdk6 with D-type cyclins. Cdk2/cyclin E complexes function later in G1 and are required for the G1 to S transition. Cdk2/cyclin A complexes are then required for progression through S phase, and Cdc2/cyclin B complexes drive the G2 to M transition.

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10 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. S-Cdk and M-Cdk.

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12 Regulation of cell cycle involves 1. Controlled expression and destruction of cyclins, example??? 2. Activating and inhibitory phosphorylation and dephosphorylation of the CDKs, example??? and 3. Expression and destruction of inhibitory proteins that associate with CDKs, or CDK/cyclin complexes

13 Cell-Cycle Control: Transcriptional Regulation Cell-cycle control may depend exclusively on post-transcriptional mechanisms that involve the regulation of Cdk activity by phosphorylation and the binding of regulatory proteins such as cyclins, which are themselves regulated by proteolysis (e.g. in frog embryonic cell cycle). In the more complex cell cycles of most cell types, however, transcriptional control provides an added level of regulation. Cyclin levels are controlled not only by changes in cyclin degradation but also by changes in cyclin gene transcription and cyclin synthesis.

14 DNA replication check point (S-Cdks) Evidence from cell-fusion experiments for a rereplication block (A) S-phase cytoplasm contains factors that drive a G1 nucleus directly into DNA synthesis. (B) A G2 nucleus, having already replicated its DNA, is refractory to these factors. (C) Fusion of a G2 cell with a G1 cell does not drive the G1 nucleus into DNA synthesis, indicating that the cytoplasmic factors for DNA replication that were present in the S-phase cell disappear when the cell moves from S phase into G2.

15 Initiation of DNA replication by S-Cdks DNA replication begins at origins of replication, which are scattered at various locations in the chromosome Origin recognition complex (ORC) binds to replication origins throughout the cell cycle and serve as landing pads for several additional regulatory proteins. e.g. Cdc 6 Cdc 6 is present at low levels during most of the cell cycle but increases transiently in early G1. Cdc 6 binds to ORC at replication origins in early G1 (where it is required for the binding of a complex composed of a group of closely related proteins, the Mcm proteins). Mcm ring complexes then assemble on the adjacent DNA. Minichromosome maintenance (Mcm) proteins are a series of closely related proteins that are components of the prereplicative complex. The Mcm proteins are essential for initiating eukaryotic DNA replication and serve as useful markers of proliferating cells.

16 The resulting large protein complex is known as the prereplicative complex, or pre-rc. After binding of pre-rc, the replication origin is ready to fire. The activation of S-Cdk in late G1 pulls the trigger and initiates DNA replication. The initiation of replication also requires the activity of a second protein kinase, which collaborates with S-Cdk to cause the phosphorylation of ORC. The S-Cdk also blocks rereplication by causing the dissociation of Cdc6 from origins, its degradation, and the export of all excess Mcm out of the nucleus. Cdc6 and Mcm cannot return to reset an ORC-containing origin for another round of DNA replication until M-Cdk has been inactivated at the end of mitosis Ubiquitylation of cdc6 by SCF

17 The initiation of DNA replication once per cell cycle.

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19 DNA replication checkpoint In case a cell is driven into mitosis before it has finished replicating its DNA??? This disaster is avoided in DNA replication checkpoint (mechanism, which ensures that the initiation of mitosis cannot occur until the last nucleotide in the genome has been copied) Broken or incomplete sets of chromosomes to its daughter cells Sensor mechanisms, of unknown molecular nature, detect either the unreplicated DNA or the corresponding unfinished replication forks and send a negative signal to the cell-cycle control system, blocking the activation of M-Cdk. Normal cells treated with chemicals Hydroxyurea (inhibitors of DNA synthesis)???? Caffeine (Defective checkpoint mechanism) Cells plunge into a suicidal mitosis despite failure to complete DNA replication

20 DNA replication checkpoint Hydroxyurea blocks DNA synthesis. This block activates a checkpoint mechanism that arrests the cells in S phase, delaying mitosis. If caffeine is added as well as hydroxyurea, the checkpoint mechanism fails, and the cells proceed into mitosis according to their normal schedule, with incompletely replicated DNA. As a result, the cells die.

21 Cell-Cycle Progression is Blocked by DNA Damage: DNA damage checkpoints When chromosomes are damaged, as can occur after exposure to radiation or certain chemicals, it is essential that they be repaired before the cell attempts to duplicate or segregate them. Most cells have at least two such checkpoints 1. Chk1 (In late G2, which prevents entry into S phase), and 2. Chk2 (In late G1, which prevents entry into mitosis).

22 ATR Kinase (single stranded lesion) ATR phosphorylates and activates a check point kinase Chk1 Chk1, in turn phosphorylates Cdc25 on serine residue (making it a target for a special adaptor protein that binds to phosphatase in the cytoplasm) Result is the inhibition of Cdc25 s phosphatase activity and prevents from being reimported to nucleus) Importance of cdc25 in cell cycle Absence of Cdc25 from the nucleus leaves the cdk in an inactive state---- Cell Cycle Arrests at G2 ATM kinase (double stranded nick) ATM phosphorylates and activates a check point kinase Chk2 Chk2, in turn phosphorylates a transcription factor p53 This activation leads to transcription and translation of p21 gene (cdk inhibitor that arrests cells in G1 until damage repaired) P21 subsequently leads to inhibition of Cdk Cell Cycle Arrests at G1

23 Cell cycle arrest

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25 If ATM or ATR gene is mutated? If p53 gene is mutated? If p21 gene is mutated?

26 Entry into Mitosis:Activation of M-Phase Cyc-Cdk Complexes Completion of DNA replication leads to M phase The events of mitosis are triggered by M-Cdk/MPF Duplicated chromosomes and other cell contents are distributed equally to the daughter cells The activation of M-Cdk begins with the accumulation of M-cyclin. In embryonic cell cycles, the synthesis of M-cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from a decrease in its degradation. In most cell types, M-cyclin synthesis increases during G2 and M, owing primarily to an increase in M-cyclin gene transcription leading to a gradual accumulation of M-Cdk as the cell approaches mitosis.

27 MPF: M-phase or Maturation Promoting Factor MPF was first isolated from the cytoplasm of maturing xenopus oocytes. Triggers passage through G2 checkpoint into M-phase The concentration of MPF was found to vary dramatically during oocyte development. Injection of cytoplasm or purified MPF into oocytes arrested in G2 caused the oocytes to proceed through mitosis.

28 Identification of MPF

29 MPF: Composition MPF is composed of two key subunits: Cdc2 and Cyclin B. Cdc2 is the protein that is encoded by genes which are required for passage through START as well as for entry into mitosis (in yeast). Cyclin B is a regulatory subunit required for catalytic activity of the Cdc2 protein kinase. In Humans cdc2=cdk1 START=Restriction point

30 Structure of MPF

31 MPF activity is dependent upon Cyclin B Accumulation and degradation of cyclins

32 MPF regulation Cdc2 forms complexes with cyclin B during S and G2. Cdc2 is then phosphorylated on threonine-161 by CAK, which is required for Cdc2 activity, as well as on tyrosine-15 (and threonine-14 in vertebrate cells) by wee1, which inhibits Cdc2 activity. Dephosphorylation of Thr14 and Tyr15 activates MPF at the G2 to M transition. MPF activity is then terminated toward the end of mitosis by proteolytic degradation of cyclin B.

33 Regulation of MPF

34 The activation of M-Cdk In case of mutation in Wee1 or cdc25?

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37 Activation of M-cdk complex 1. Activation of cdc25 protein in late G2 Two protein kinases activate cdc25 1. Polo Kinase 2. M-cdk itself (Activation of its own activator) 2. Suppression of Wee1 M-cdk (Suppression of own suppressor)

38 Feedback mechanism of M-Cdk

39 Separation of Duplicated Chromosomes: M-Cdk M-Cdk must induce the assembly of the mitotic spindle and ensure that replicated chromosomes attach to the spindle. M-Cdk also triggers chromosome condensation, nuclear envelope breakdown, actin cytoskeleton rearrangement, and the reorganization of the Golgi apparatus and endoplasmic reticulum. Triggered through phosphorylation of specific structural or regulatory proteins by M-cdk. The breakdown of the nuclear envelope, requires the disassembly of the nuclear lamina (the underlying shell of polymerized lamin filaments that gives the nuclear envelope its structural rigidity). Direct phosphorylation of lamin proteins by M-Cdk results in their depolymerization, which is an essential first step in the dismantling of the envelope.

40 Chromosome condensation Phosphorylation by M-Cdk. Condensin complex (A complex of 5 proteins), is required for chromosome condensation in Xenopus embryos. Phosphorylated subunits in the complex, are able to change the coiling of DNA molecules. (thought to be important for chromosome condensation during mitosis) Phosphorylation by M-Cdk also triggers the complex microtubule rearrangements and other events that lead to the assembly of the mitotic spindle.

41 Separation of Sister Chromatids: By Proteolysis After entry into mitosis, the cell cycle reaches its culmination with the separation of the sister chromatids at the metaphase-to-anaphase transition. APC initiates sister-chromatid separation Cohesion complex is involved in sister-chromatid cohesion The cohesin proteins (COHESINS) are closely related to the proteins of the condensin complex involved in chromosome condensation Anaphase begins with the disruption of the cohesion between sister chromatids, through activation of the APC enzyme complex. The target of the APC is the protein SECURIN. Before anaphase, securin binds to and inhibits the activity of a protease called SEPARASE. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of the cohesin complex. In an instant, the cohesion complex falls away from the chromosomes, and the sister chromatids separate.

42 The triggering of sister-chromatid separation by the APC

43 Experiments demonstrating the requirement for protein degradation to exit from mitosis (A) An APC inhibitor was added to frog egg extracts undergoing mitosis (B) A non-degradable mutant form of M-cyclin was added to mitotic frog egg extracts (arrested mitosis after sister-chromatid separation) Indicating that destruction of M-cyclin is not required for sister-chromatid separation but is required for the subsequent exit from mitosis.

44 If APC triggers anaphase, what triggers APC? APC activation requires the protein Cdc20, which binds to and activates the APC at mitosis. At least two processes regulate Cdc20 and its association with the APC. 1. Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene (Transcriptional regulation) 2. Phosphorylation of APC helps Cdc20 bind to the APC, thereby helping to create an active complex (It is not clear what kinases phosphorylate and activate the Cdc20-APC complex) M-Cdk activity is required for the activity of these kinases, but there is a significant delay, or lag phase, between M-Cdk activation and the activation of the Cdc20-APC complex. The molecular basis of this delay is still mysterious, but it is likely to hold the key to how anaphase is initiated at the correct time in M phase.

45 Control of Proteolysis by APC

46 Unattached Chromosomes Block Sister-Chromatid Separation: The Spindle-Attachment Checkpoint In most cell types, a spindle-attachment checkpoint mechanism operates to ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation occurs. The checkpoint depends on a sensor mechanism that monitors the state of the kinetochore (specialized region of the chromosome that attaches to microtubules of the spindle). Any kinetochore that is not properly attached to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister-chromatid separation. The nature of the signal generated by an unattached kinetochore is not clear. Sister-chromatid separation cannot occur until the last kinetochore is attached.

47 Exit from Mitosis: Inactivation of M-Cdk After the chromosomes have been segregated to the poles of the spindle, the cell must reverse the complex changes of early mitosis spindle must be disassembled, chromosomes decondensed, and nuclear envelope reformed. Phosphorylation of various proteins is responsible for getting cells into mitosis in the first place, Dephosphorylation of the same proteins is required to get them out In principle, these dephosphorylations and the exit from mitosis could be triggered by The inactivation of M-Cdk, The activation of phosphatases, or Both these events can occur (Of primary importance) Ubiquitylation of the cyclin is usually triggered by the same Cdc20- APC complex that promotes the destruction of Securin at the metaphase-to anaphase transition The activation of the Cdc20-APC complex leads not only to anaphase, but also to M- Cdk inactivation which in turn leads to all of the other events that take the cell out of mitosis.

48 Control of Proteolysis by APC

49 The G1 Phase: A State of Stable Cdk Inactivity In early animal embryos, the inactivation of M-Cdk in late mitosis is due almost entirely to the action of Cdc20-APC. The destruction of M-cyclin (A stimulator of APC) in late mitosis leads to the inactivation of all APC activity in an embryonic cell, allowing the cell to quickly begin accumulating new M-cyclin for the next cycle. Rapid cyclin accumulation immediately after mitosis is not useful in cell cycles containing a G1 phase, which allows cell growth as well as cell cycle regulation by extracellular signals.

50 Mechanisms to prevent reactivation of CDK after mitosis 1. Use of another APC-activating protein called Hct1, a close relative of Cdc20 Although both Hct1 and Cdc20 bind and activate the APC, they differ in one important respect. Cdc20-APC complex is activated by M-Cdk Hct1-APC complex is inhibited by M-Cdk (phosphorylates Hct1) As a result of this relationship, Hct1-APC activity increases in late mitosis after the Cdc20-APC complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20-APC activity has declined, Hct1-APC activity is high.

51 2. Increased production of CKIs Budding yeast cells contain a CKI protein called Sic1, which binds to and inactivates M-Cdk in late mitosis and G1. Like Hct1, Sic1 is inhibited by M-Cdk, which phosphorylates Sic1 during mitosis. M-Cdk also phosphorylates and inhibits a gene regulatory protein required for Sic1 synthesis, resulting in decreased Sic1 production. Sic1 and M-Cdk, like Hct1 and M-Cdk, mutually inhibit each other resulting in decline in M-Cdk activity that occurs in late mitosis triggers the rapid accumulation of Sic1 protein, and this CKI helps ensure that M-Cdk activity is stably inhibited after mitosis. 3. Decrease in cyclin production

52 Escape from stable G1 to S phase: Accumulation of G1-cyclins In budding yeast Cyclins are not targeted for destruction by Hct1-APC and are not inhibited by Sic1. Accumulation of G1 cyclins leading to an increase in G1-Cdk activity G1-Cdk activity triggers the transcription of G1/S-cyclin genes Increased synthesis of G1/S-cyclins and the formation of G1/ S-Cdk G1/S-Cdk activity commits the cell to enter S phase It also stimulates the transcription of S-cyclin genes, leading to the synthesis of S-cyclins and the formation of S-Cdk complexes. G1/S-Cdk phosphorylates and inactivates Sic1 and Hct1-APC (FEED BACK) In animal cells Stimulated by the extracellular signals that promote cell proliferation

53 The control of G1 progression by Cdk activity in budding yeast. As cells exit from mitosis and inactivate M-Cdk, the resulting increase in Hct1 and Sic1 activities results in stable Cdk inactivation during G1. When conditions are right for entering a new cell cycle, the increase in G1-Cdk and G1/S-Cdk activities leads to the inhibition of Sic1 and Hct1 by phosphorylation, allowing S-Cdk activity to increase.

54 Brake in Mammalian G1 Cells: The Rb Protein Animal cells suppress Cdk activity in G1 by Hct1 activation, The accumulation of a CKI protein (p27 in mammalian cells), and Inhibition of cyclin gene transcription Activation of G1-Cdk complexes Mediated by a gene regulatory protein called E2F. E2F binds to specific DNA sequences in the promoters of genes encoding proteins required for S-phase entry, including G1/S-cyclins and S-cyclins. E2F function is controlled primarily by an interaction with the retinoblastoma protein (Rb), an inhibitor of cell-cycle progression. During G1, Rb binds to E2F and blocks the transcription of Sphase genes. When cells are stimulated to divide by extracellular signals, active G1-Cdk accumulates and phosphorylates Rb, reducing its affinity for E2F The Rb then dissociates, allowing E2F to activate S-phase gene expression

55 RB and E2F are associated in an inactive complex, late M phase through middle of G1. Late G1: cdk2/cyclin A complex forms and Rb is phosphorylated. Rb-P can no longer bind E2F E2F is a transcription factor that can now activate transcription of genes required for DNA synthesis, or S phase.

56 The transcriptional control system includes feedback loops that sharpen the G1/S transition 1. The liberated E2F increases the transcription of its own gene 2. E2F-dependent transcription of G1/S-cyclin and S-cyclin genes leads to increased G1/S-Cdk and S-Cdk activities, which in turn increase Rb phosphorylation and promote further E2F release 3. The increase in G1/S-Cdk and S-Cdk activities enhances the phosphorylation of Hct1 and p27, leading to their inactivation or destruction

57 E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop. Mechanisms controlling S-phase initiation in animal cells G1-Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation (inactivates Rb, freeing E2F to activate the transcription of S-phase genes and the genes for G1/S-cyclin and S-cyclin G1/S-Cdk and S-Cdk activities further enhances Rb phosphorylation, forming a positive feedback loop.

58 prb: Guardian of R Point After passing R point, cell cycle is an autonomous program. It will advance to be finished, if no DNA damage prb) is also called pocket protein. Three isoforms: prb, p107, and p130.

59 p53 activates p21 p21 inhibits cyclin/cdk2 activity p53 mutant in > 50% of cancers Blocks cell cycle progression and initiates cell death!

60 An overview of the cell-cycle control system

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