5.3. Cell division cycle regulation :
Regulation of cell cycle is crucial to ensure proper segregation of genetic material for the normal development and maintenance of multicellular organisms. Failure of which leads to genetic instability and uncontrolled cell division. The complex macromolecular events of the cell cycle are regulated by a series of cellular proteins. The key regulatory protein of cell division cycle is a heteromer consist of Cyclin and Cyclin Dependent protein Kinase (CDK), a family of serine–threonine protein kinases that are activated at specific points of the cell cycle. Cyclin is a regulatory subunit and CDK is catalytic subunit.
Cyclin Dependent Kinase (CDKs)
CDks are kinase that transfer the phosphate group to other protein substrates. Phosphorylation cause change in the enzymatic activity of the substrate to interact with other proteins. CDKs are activated by binding to its regulatory subunit cyclins. Nine CDK have been identified, of which five remains active during cell cycle, i.e. during G1 (CDK4, CDK6 and CDK2), S (CDK2), G2 and M (CDK1). Throughout the cell cycle, level of cyclins increases and decreases thus periodically activate different CDKs. The cyclin-CDK complex is formed as a result of the interaction between the cyclin box, a region conserved across cyclins and a region of the CDK, called the PSTAIRE.
Different cyclins are required at different stages of the cell cycle. Cyclin-CDK complex ( cyclin D1,D2,D3 binds with CDK4 and CDK6) is necessary for entry in G1. Cyclin E also associates with CDK2 to regulate progression from G1 into S phase. Cyclin A and CDK2 complex is required during S phase. In late G2 and early M, cyclinA- CDK1 promotes the entry into M. Mitosis is further regulated by cyclinB – CDK1.
Cyclins are family of proteins that binds with the regulatory subunit of CDK and make them active. Cyclin was originally named so because their concentration varies in a cyclic fashion during the cell division cycle.
Types of Cyclins
Classification of Cyclin is based upon the conserved cyclin box structure. The fluctuation in cyclin concentration during the cell cycle is because of destruction of cyclin by ubiquitin proteasome system. Cyclin has no catalytic activity. Different cyclins are required at different stages of the cell cycle.
Cyclin D synthesis is initiated during G1 and drives the G1–S phase transition. When growth factors and mitogens or nutrients (for unicellular organism) are present, cells start expression of Cyclin D. Growth factors works through RTK pathways and stimulate the Ras Raf ERK . This leads to the activation of Myc. Myc increase the rate of transcription of cyclin D. Cyclin D initiate the cell cycle and it interacts with two CDKs, CDK4 and CDK6.
The Cln3 is a yeast homologue of cyclin D. It interacts with Cdc28 (cell division control protein) during G1. In proliferating cells because of continuous signaling by growth factor, the cyclin D-CDK4/6 complex start accumulation. This accumulation is of great importance for cell cycle progression. Cyclin D-CDK4/6 complex partially phosphorylates retinoblastoma tumor suppressor protein (Rb). This phosphorylation of Rb is important for S phase progression. The phosphorylation of RB causes dissociation of RB from E2f. E2f is a transcription factor for CyclinE. Cyclin E is important for S phase progression.
The transition from G1 to S phase requires binding between Cyclin E to G1 phase CDK2. The Cyclin E–CDK2 complex phosphorylates p27Kip1 . p27Kip1 an inhibitor of Cyclin D. Cyclin D remain unbounded form as the cyclin E is already bounded with CDK2. The phosporylated form of p27Kip1 is tagged by ubiquitin and get degraded by UPS. Thus promoting expression of Cyclin A, allowing progression to S phase.
Cyclin A plays a role in the regulation of two different stages of cell cycle. It can associate with CDK2 and CDK1.Association of cyclin A with CDK2 is required for passage into S phase while association with CDK1 is required for entry into M phase. In S phase of cell division cycle Cylin A-CDK2 complex is present.
Cyclin B is a mitotic cyclin. The complex of CDK and cyclin B is called maturation promoting factor or mitosis promoting factor (MPF). Cyclin B is necessary for the progression of the cells into and out of M phase of the cell cycle. Cyclin B synthesis starts in S phase but it is exported out of Nucleus because it has nucleus exporting sequence. Cyclin B-CDK1 complex activation causes breakdown of nuclear envelope and initiation of prophase and deactivation of this complex leads the cell to exit mitosis.
During late Anaphase stage cyclin B concentration fall abruptly due to degradation by UPS (Cdk1 is constitutively present).
Cyclin A and cyclin B have destruction box and cyclin D and E contains a PEST [P:Proline, E:glutamic acid, S:serine, T:threonine] sequence required for efficient ubiquitin-mediated cyclin proteolysis at the end of phase of the cell cycle. Ubiquitin mediated proteolysis of cyclin is catalysed by ubiquitin ligase enzyme. Skp1-Cullin-F-box (SCF) and anaphase promoting complex (APC) are responsible for the ubiquitylation and destruction of cyclins.
Regulation of CDK
CDK activity can be regulated by three mechanisms
- Synthesis and degradation of cyclins.
- Phosphorylation of conserved threonine and tyrosine residues.
Wee1 and Myt1 are protein kinase which phosphorylates the CDK1 at tyrosine-15 and/or at threonine-14. Phosphorylation inhibit the activity of CDK1. Cdc25 phosphatase dephosphorylates the sites and this leads activation of CDK1.
Dephosphorylation at these sites are necessary by the enzyme Cdc25 phosphatase for activation of CDK1 and further progression through the cell cycle. Full activation of the cyclin-CDK complex occurs when CDK7-cyclinH complex(CDK-activating kinase), phosphorylates threonine 161 (threonine 172 in CDK4 and threonine 160 in CDK2) residue near the CDK active site.
3. By binding with inhibitors
CDK activity can also be regulated by cell cycle inhibitory proteins, called CDK inhibitors(CKI) which binds to CDK alone or to the CDK-cyclin complex. Two distinct families of CKI have been discovered.
- The INK4 family- P15 INK4b, P16 INK4a, P18 INK4c , P19 INK4d which inactivates G1 CDK (CDK4 and CDK6).
- The Cip/Kip family- P21 Waf1, Cip1, P27Cip2, P57Kip2 which inhibits the cyclinA-CDK2 activity.
Both the internal and external signals are responsible for the regulation of CKI.
For example: p53 (tumor supressor gene) controls the expression of P21. The P21 gene promoter consists of a P53 binding site which allows P53 to transcriptionally activate the P21 gene. The expression of P15 and P27 increases and as a result the cells get G1 arrested as the P15 and P21 binds to CDK4 and arrest the cell cycle in G1 phase.
The intracellular localization of cell cycle proteins also contributes to a proper cell cycle progression. Until the beginning of prophase stage, cyclin B having a nucleus exclusion signal is exported outside the nucleus.
During early G1 , pRb (the product of retinoblastoma tumor suppressor gene) phosphorylates resulting in the disruption of the complex with histone deacetylase (HDAC) and transcription factors like E2F-1 and DP-1 are released that are required for the progression of S phase. pRb remains in hyperphosphorylated form for the rest of the cycle. During G1 -S transition, inhibitor p27 is phoshorylated by CDK2 and cyclin E complex associated with proteasome degradation. CDK2 and cyclin E also phosphorylates NPAT (Nuclear protein mapped to ATM locus), high levels of which plays a crucial role in S phase entry. CDK2-cyclinE phosphorylate histone H1 required during DNA replication for chromosome condensation. Some of the CDK substrates include Wee1 and cdc25, nuclear lamins,vimentin, and other cytoskeletal proteins required for proper mitosis.
Phosphorylation of Rb allows E2F-DP to dissociate from pRb and become active. When E2F is free it activates factors like cyclins (e.g. Cyclin E and A).
Role of Rb/E2F pathway in cell cycle :Retinoblastoma is a tumor caused from the immature cells of retina in children. It occurs due to the presence of inactivated copies of Rb gene , a tumor suppressor gene. Normally, the Rb protein interacts with the E2F transcription factor preventing it from interacting with the cell machinery. While, in the absence/defect of pRb, E2F (binds with DP) induces the transactivation of E2F gene which encodes proteins involved in DNA replication (DNA polymerase, thymidine kinase, dihydrofolate reductase, PCNA)and chromosome replication. Thus, facilitating the G1 -S transition and S phase. In homozygous Rb- / Rb- cells, Rb is permanently inactive. Therefore, E2F promotes S phase and the duration of G1 is reduced resulting in the formation of tumor.
During the M-to-G1 transition, pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state Rb.
5.4. Cell Cycle Checkpoints
Cell cycle is a highly regulated sequential process, monitored by a number of checkpoints. Checkpoints controls function to ensure that incomplete or damaged chromosomes are not replicated and that critical stages of cell cycle are completed before the following stages is initiated.
The eukaryotic cell cycle is guarded mainly by three checkpoints:
- G1 -S checkpoint
- G2 -M checkpoint and
- Metaphase/anaphase checkpoint (spindle assembly checkpoint).
5.4.1. G1 - S Check point or restriction or start checkpoint
DNA is replicated in S phase. The Cell cycle arrests in G1 phase if DNA is damage. DNA damage is because of irradiation with UV light or γ rays or any chemical modification.
Under normal conditions, p53 (transcriptional factor)is extremely unstable, so its level is low in cell but damage DNA leads to an increase in its concentration. The p53 stimulates the transcription of various genes like p21 (CIP), Mdm and Bax (apoptotic genes). p21 is CKI. It inhibits CDK and prevents the replication of damaged DNA and arrest the cell in G1 stage. The mdm protein work as E3 ligase for p53. P53 increase the mdm protein expression which in turn degrade p53, thus providing a negative loop. The proteasomal degradation of p53 through polyubiquitination by a ubiquitin ligase Mdm results in low transcription activity of p53, hereby providing a negative loop. In case of extensive DNA damage, p53 induces cell death by activating genes (Bax, Fas) that are involved in apoptotic signaling.
Different protein kinase like ataxia-telangiectasia-mutated(ATM), ataxia and rad3 related(ATR) detects DNA damage. These kinases phosphorylates p53, thereby increases the transcription of p21 resulting in blocking the cell cycle at the G1 /S checkpoint.
In G1–S transition, the all mitochondria of a cell fused to form a giant, single tubular network. This network is electrically coupled and the membrane potential (ψm) of mitochondria is hyper polarized. Thus allowing generation of more energy during cell division.
The G1 – S checkpoint is controlled by many different stimuli including (1) DNA damage (2) TGFβ (3) Contact inhibition (4) Replicative senescence, and 5. Growth factor withdrawal.
Transforming growth factor beta (TGF-β) controls proliferation and cellular differentiation in cells. TGF-beta acts as an antiproliferative factor in normal epithelial cells. TGF-β is a secreted protein that exists in three isoforms called TGF-β1, TGF-β2 and TGF-β3.
TGFβ prevents phosphorylation of RB scheduled in mid to late G1 and arrests cells in late G1. TGF β activates SMAD proteins and SMAD activates CKI.
When TGF-beta1 binds to a TGF receptor and activates SMAD3-SMAD4. SMAD activates cyclin-dependent kinase inhibitors (p16, p15, p21, p27). TGF β additionally inhibits the transcription of Cdc25A, a phosphatase that activates the cell cycle kinases. For this reason cell cycle kinases (CDK2, CDK4, CDK6) bound to Cyclin E, and Cyclin D cannot phosphorylate pRb. Cdc 25A is a phosphatase that activates CDK.
5.4.2. DNA Damage
The p53 is inactive in normal cell and it is bound to MDM2 protein. MDM2 is a E3 ligase which degrades the P53. The P53 is activated by UV, oncogenes and drugs or other substances that damage DNA.
Damage of DNA activates ATM, Chk1 and Chk2. Which phosphorylate p53 and prevent the binding of p53 with MDM2. Once activated P53 work as transcription factor for P21.which binds the complex G1-S/CDK and D / CDK (molecules important for the transition from G1 to S phase) by inhibiting their activity ( and avoiding the proliferation of mutated cells). p53also inhibits angiogenesis (formation of new blood vessel).
Activation of proliferation : many grow factors are involved in proliferation like
IGF1 and FGF2
Insulin Growth Factor-I and Fibroblast Growth Factor-2 both enhance cyclin D1 and cyclin E-cdk2 association. This activity help in G1 progression. IGF-I was required for G(2)/M progression. FGF-2 also decrease the levels of the cdk inhibitor p27(Kip1) associated with cyclin E-cdk2.
PDGF induces the formation of cyclinD-cdk4 complexes. This complexes binds with P27. PDGF also reduce the level of free P27.
pRb (Retinoblastoma Protein)
The retinoblastoma protein is a tumor suppression protein. Rb is expressed throughtout the cell cycle but it is phosphotylated during the G1/S transition.RB play a role in G1 arrest when the DNA is damage. Rb play a role in G1 arrest.
The unphosphortlated form of RB is Antiproliferatic. Its inhibits the transcription of genes that are required for cell cycle progression, e.g., cyclin A.
Rb binds with E2F family of proteins. The transcription activating complexes of E2 promoter-binding–protein-dimerization partners (E2F-DP) can push a cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The pRb-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin. Because histone deacetylase modifies chromatin to a closed state through deacetylation, transcription is repressed. The antimitogenic activity of TGFb also requires RB activation.
When a quiescent cells are stimulated to enter the cell cycle, Cdk4/6-cyclin D complexes become active in mid-G1 and initiate the phosphorylation of RB. Later in G1, RB becomes hyperphosphorylated through the combined actions of Cdk4-cyclin D, Cdk2-cyclin E, and Cdk2-cyclin A. The activities of Cdk2-cyclin E and Cdk2-cyclin A are both rate limiting and required for entry into S phase. Phosphorylation of RB is maintained throughout S and G2, until RB is finally dephosphorylated by a phosphatase at the M/G1 transition.
Phosphorylation of pRb allows E2F-DP to dissociate from pRb and become active. When E2F is free it activates factors like cyclins (e.g. Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA.
The amplification or overexpression of cyclin D and Cdk4/6, or loss of the Cdk4/6 inhibitor P16INK4a causes increased RB phosphorylation and inactivation of RB function and this leads to tumors.
Phosphorylation Site-Mutated RB proteins (PSM – RB)
Phosphorylation Site-Mutated RB proteins are those mutant form of RB which cannot be phosphotylated by Cdk. These PSM – RB remains in bound form with E2F and block the cell cycle. These (PSM-RB) cause a cell cycle arrest in G1.
5.4.3. G2 -M checkpoint
Prevents the mitotic entry in response to DNA damage. DNA damage induces the ATM/ATR pathway which activates the Chk1 and Chk2 kinases. Chk1/2 phosphorylates Cdc25 inhibiting its activity and promotes its binding to 14-3-3 proteins to sequester it in the cytoplasm. p53 also activates p21, and both p21 and 14-3-3 proteins further inhibit CDK1-cyclin B complex by phosphorylation and sequestering cdc2 in the cytoplasm. p53 is also involved in the dissociation of CDK1-cyclin B complexes by induction of Gadd45 (growth damage and DNA damage inducible gene).
5.4.4. Spindle fibre checkpoint
Proper chromosome segregation is crucial to maintain genomic integrity. The spindle is made up of a bipolar array of microtubules (MTs) extending from opposing spindle poles to chromosomes at the spindle equator. Microtubules are polar polymers with the “minus” ends located at spindle poles and “plus” ends free to attach to chromosomes. Chromosomes are connected to spindle microtubules via kinetochores.
Spindle fibre checkpoint examines improper alignment of the chromosome on the mitotic spindle via kinetochores and thereby determines whether to execute or delay chromosome segregation. The major components involved in spindle checkpoint are Mad1/2/3 (mitotic arrest deficient), (Budding Inhibited by Benzimidazole) (Bub1/2) respectively. These proteins are activated when improper attachment of microtubule occurs and inhibit the Cdc20 subunit of the anaphase-promoting complex (APC) and as a result metaphase anaphase transition is prevented.
5.5. Anaphase promoting complex (APC)
Anaphase Promoting Complex is a multisubunit E3 ubiquitin ligase that plays a critical role in cell cycle by inducing proteolysis of different cell cycle regulators. APC or cyclosome is a large protein complex with 12-13 core components that remain stably associated. Multisubunit complex includes SCF and several SCF-like complexes containing a RING (APC11)subunit, a cullin (APC2) subunit.
The major function of APC is to induce transition from metaphase to anaphase by tagging specific proteins for proteolysis. Securin and S and M cyclins are the two basic proteins that gets degraded as substrates of APC/C. During metaphase, cohesin mediates the binding of the sister chromatids together. But when securin undergoes ubiquitination, releases separase which further triggers the degradation of cohesin. This makes the sister chromatids free to move to opposite poles for the onset of anaphase stage. The APC/C also degrades the mitotic cyclins, resulting in the inactivation of M-Cdk complexes and thus promoting exit from mitosis and cytokinesis.
5.5.1. Regulation of sister Chromatid separation by the APC
Proteolysis of securin mediated by APC-C CdC-20 at metaphase liberates separase, a protease that cleaves the SCC1 cohesion subunit responsible for sister chromatid cohesion. APC-C CdC-20 also initiates cyclin B degradation in mitosis, which is important for activation of separase. Activation of APC-C CdC-20 is controlled by Emi1 and the spindle checkpoint. Securin degradation not only promotes sister chromatid separation but also releases the phosphatase Cdc14 from the nucleolus. The Cdc14 protein is part of a complex mitotic exit network. Release of Cdc14 leads to dephosphorylation and activation of Cdh1. Activated APC-C Cdh–1 finally degrades mitotic cyclin. Further at the M to G1 transition and maintains their low state of activity during G1
APC–C consists of a catalytic core which includes the cullin subunit Apc2 and RING H2 domain subunit Apc11. The domain of Apc2 forms a tight complex with Apc11,and mediates the ubiquitylation of substrates. The other core proteins providing molecular scaffold support includes Apc1, largest subunit and Ap). APC–C substrates have recognition amino acid i.e. can have D-box sequence (RXXLXXXXN) or KEN-box sequence (KENXXXN) that enable the APC/C to identify them where R represents arginine, L is leucine, N is asparagine, K is lysine, E is glutamate and X is any amino acid.
5.5.2. Regulation of APC activity
The APC is active from mitosis until the end of G1.The affinity of activators of the APC is regulated by phosphorylation of APC subunits. Cdc20 activity is regulated by transcription and APC-C Cdh–1 dependent degradation leading to APC-C Cdh–20 activity from early mitosis until the M to G1 transition . Cdk1 and MAPK can phosphorylate Cdc20; although it is controversial whether phosphorylation of Cdc20 is necessary for APC activation in human cells, it is required for its inhibition by the spindle checkpoint
Initially it has been reported that the protein level of Cdh1 remains relatively constant throughout the cell cycle) and Cdh1 is activated by dephosphorylation from the M to G1 transition until the end of the G1 phase. However, there are also reports that Cdh1 levels oscillate during the cell cycle at least in human cells. The subcellular localization of Cdh1 is cell cycle regulated and Cdh1 is nuclear during G1 and in the cytoplasm between S phase and the end of mitosis. Cdk-dependent phosphorylation leads to efficient inactivation of Cdh1 by nuclear export and prevention of APC binding, thus restricting APCCdh1 activity to the points in the cell cycle when Cdk activity is low.
Phosphorylation of core APC subunits upon entry into mitosis by Cdk1/cyclin B and polo protein kinases Plk/Cdc5/Plo1 enhances Cdc20–APC interaction. One or more subunits (Apc1, Cdc27, Cdc16 and Cdc23are phosphorylated during mitosis ) and dephosphorylation can inactivate the mitotic APC. Dephosphorylation of Cdk1-phosphorylation sites of the APC may inactivate the mitotic APCCdc20 by dissociation of Cdc20, and Cdh1 then replaces Cdc20, leading to degradation of Cdc20 during mitotic exit and G1. On the other hand, protein kinase A (PKA)-mediated phosphorylation of core APC subunits can also inhibit APC activityand dephosphorylation of PKA phosphorylation sites, possibly by the phosphatases (PPs) PP1 or PP2A increases APC activity and Cdc20 binding. Therefore, the phosphorylation of some subunits may have activating effects, whereas the phosphorylation of other subunits may have inhibitory effects. However, the precise nature of these phosphorylation/dephosphorylation events and how they affect APC regulation still remains to be elucidated in more detail.
5.6. Metaphase to anaphase transition:
Model of the regulation of mitosis and G1 by APC-dependent cyclin proteolysis. The first phase of cyclin B1/Clb2 proteolysis by APCCdc20 mediates spindle disassembly and cytokinesis. APCCdc20-dependent degradation of cyclin A2/Clb5 leads to activation of Cdh1 and in yeast of the CKI Sic1 downregulating mitotic kinase activity in G1. Whether there is any role of human CKIs such as p21 or p27 in this context has to be defined. APCCdh1 inactivates cyclin B1/Clb2 further during mitotic exit and the G1 phase of the cell cycle to regulate cell growth and the length of G1 allowing cell differentiation and correct assembly of pre-RCs at origins of replication and subsequent complete and accurate DNA replication
Metaphase Cyclin A is degraded and at the termination of mitosis Cyclin B is degraded.
Regulation of cell cycle progression by APC/C-dependent proteolysis. (A) Sister chromatid separation and Cdk1 inactivation both depend on APC/CCdc20. Activation of APC/CCdc20 but not APC/CCdh1 requires phosphorylation of core subunits by Cdk1–cyclin B. APC/CCdc20 mediates sister chromatid separation and Cdk1 inactivation by degrading securins and B-type cyclins, respectively. APC/CCdc20also creates the conditions that keep Cdk1–cyclin B complexes inactive during the ensuing G1 phase. Cdk1 inactivation by APC/CCdc20 permits release from its inhibitor of the Cdc14 phosphatase which, by dephosphorylating Sic1 and Cdh1, causes accumulation of a CKI and activation of APC/CCdh1. Broken lines indicate proteolysis and P in a circle indicates phosphorylation. (B) Changes in the abundance and activity of CDKs, the securin Pds1, the CKI Sic1, and both forms of the APC/C during the cell cycle.
Anaphase is the stage of mitosis or meiosis when chromosomes are split and the sister chromatids move to opposite poles of the cell. During metaphase, APC/C is inhibited until all the sister kinetochores are attached to opposite poles of the mitotic spindle. When all the kinetochores are properly attached, APC/C becomes active and promotes binding to cdc20. M cyclins and securin are then targeted for degradation through ubiqutylation by APC/Ccdc20 leading to the onset of anaphase stage. Pds1p control the transition from Metaphase to anaphase.CDC20 is required to degrade the pds1p.
5.7. M to G1 transition :
After the completion of mitosis, the entry into another round of mitosis is prevented by inhibiting Cdk activity. During the transition from metaphase to anaphase, Cdh1 is phosphorylated by M-Cdk and thus prevent it from attaching to APC/C. As the M-Cdk is degraded in later stage of mitosis, cdc20 gets released and Cdh1 can bind to APC/C, thus keeping it activated for the M/ G1transition. Cdh1 is necessary for APC to degrade the Cyclin B. Degradation of Cyclin B is mark of exit of Mitosis.