Cell Cycle Progression:
Early studies of the root tips of plants identified distinct stages which define the life cycle of a cell. After introduction of radioactive nucleotides at different stages, it was observed that the cells would incorporate the radioactive nucleotides into their DNA only at certain times. Such studies culminated in the definition of cell cycle stages. Cells may exist in a state of quiescence (G0) or may enter the cell cycle. A typical cell has 24 h cell cycle which is generally defined as follows:
Cell Cycle Stages:
Cyclin Dependent Kinases:
The core components of the cell cycle regulatory machinery are a family of serine/threonine kinases called the cyclin dependent kinases (CDKs). CDK activity depends on association with regulatory subunits called cyclins which are defined by a conserved 100-amino acid residue-long sequence involved in CDK-binding and activation. While endogenous CDK levels remain relatively consistent, cyclin protein levels fluctuate dramatically in accordance with cell cycle stage. Upon stabilization of a certain cyclin, association with the appropriate CDK forms a cyclin-CDK complex in which the cyclin enhances CDK activity, and directs the complex to the appropriate target protein.
Fluctuation of Cyclin Levels
Fluctuating cyclin levels cause distinct cyclin-CDK complexes to form in a cell cycle-dependent manner. During G1, D-type cyclins (D1, D2, and D3) bind two similarly acting CDKs; CKD4 and CDK6. After the restriction point (R-point) in late G1, E-type cyclins (E1 and E2) associate with CDK2. Cyclin E-CDK2 complexes phosphorylate substrates which promote entry into S-phase, upon which A-type cyclins (A1 and A2) replace E-type cyclins to form cyclin A-CDK2 complexes. As S-phase progresses, the A-type cyclins leave CDK2 to associate with CDC2 (aka CDK1). In late G2 phase, B-type cyclins (B1 and B2) replace A-type cyclins to form Cyclin B- CDC2 complexes which trigger many of the events of mitosis.
Rapid increase of one type of cyclin just prior to cyclin-CDK complex formation occurs in accordance with rapid degradation of the preceding prominent cyclin. This fluctuation occurs through a feedback system where cyclin-CDK complexes in one phase of cell cycle activate those in the subsequent phase, and inactivate those that were active in the previous phase. After the G1 restriction point, cyclin E levels increase. During the G1/S transition, cyclin E-CDK2 complexes activate cyclin A. Cyclin A-CDK2 complexes inactivate the transcription factor which induced cyclin E levels, thus resulting in collapse of cyclin E levels upon S phase entry after cyclin A levels increase. Cyclin A levels decrease upon G2 entry after cyclin A-CDK2 complexes activate cyclin B. Thus, beyond the G1 restriction point, cyclin-CDK complex formation and activation occurs in an autonomous manner through a feedback system independent of extracellular signals. This process ensures completion of cell cycle progression after the commitment is made to cross the G1 restriction point.
Prior to the G1 restriction point, extracellular signals influence the decision for cell cycle entry through control over D-type cyclin levels. Different types of D-type cyclins are activated by distinct signalling pathways which are stimulated by different surface receptors. For example, mitogenic growth factors activate receptor tyrosine kinases on the cell surface to induce signalling cascades which ultimately result in the AP-1 transcription factor binding to and activating the cyclin D1 promoter, resulting in rapid accumulation of cyclin D1. Removal of growth factors from a cell’s medium results in an equally rapid collapse of cyclin D1 levels.
Cyclin Activating Kinases
There are currently 20 members of the Cdk family, each characterized by a conserved catalytic core made of an ATP-binding pocket, a PSTAIRE-like cyclin-binding domain, and an activating T-loop motif.
CDK activation minimally depends on 2 events: 1) Binding to a cyclin; 2) Phosphorylation of a conserved theronine residue on the activating CDK T-loop. In metazoans, CDK 7 appears to be the CDK-activating kinase (CAK) for CDK 4/6, CDK 2, and CDK 1. CDK7 forms a trimeric complex with cyclin H and the RING-finger protein Mat 1. Expression and activity of the 3-subunit CAK complex are constant throughout the cell cycle of actively proliferating cells.
CDK Inhibitors
CDK inhibitors (CdkIs) antagonize the activities of the cyclin-CDK complexes, thus are negative regulators of the cell cycle. These inhibitors are induced by antimitogenic signals. One group of CdkIs called INK4 proteins (originally inhibitors of CDK4) target the G1 cyclin-CDK complexes (CDK4 and CDK6 complexes). The four known INK4 proteins, p16INK4A, p15INK4B, p18INK4C, and p19INK4D, inhibit Cdk4/6 activity by allosteric competitionof their binding sites with cyclins. Mechanistically, INK4A proteins are known to distort both the cyclin-binding site and the ATP-binding site of CDK6 to compromise catalytic activity. The Cip/Kip group of CdkIs inhibit all other cyclin-CDK complexes which form in later stages of cell cycle, and consist of the following members: p21Cip1 (aka p21Waf1), p27Kip1, and p57Kip2. Co-crystallization reveals how one domain of p27Kip1 obstructs the ATP-binding site in the catalytic cleft of CDK2.
Extracellular signals can influence the cell cycle through control over levels and localization of the CDK inhibitors. For example, TGF-β antagonizes cell proliferation through modulation of CdkI levels. TGF-β activation of the TGF-β receptor results in increased levels of p15INK4B which inhibits cyclin D-CDK4/6 complex to prevent cell cycle progression beyond the G1 restriction point. TGF-β also weakly induces p21Cip1 which blocks activity of the remaining “non-G1” cyclin-CDK complexes. More potent induction of p21Cip1 occurs in response to physiological stresses such as DNA damage. Until the genome is repaired, p21Cip1 inhibits cyclin E-CDK2 complexes to block cell cycle advance beyond the G1 restriction point, and inhibits PCNA (proliferating-cell nuclear antigen) which is a key component of the DNA replication machinery.
In contrast to the above examples of extracellular growth-inhibitory signals, growth-promoting mitogens stimulate cell cycle progression though inhibition of CdkIs. For example, mitogens stimulate receptor tyrosine kinases which activate the phosphatidylinositol 3-kinase (PI3K) pathway, leading to activation of the Akt/PKB kinase. Akt/PKB phosphorylates p21Cip1 in the nucleus, inducing nuclear export. Akt/PKB also phosphorylates p27Kip1 to prevent nuclear import.
Both Ink4 and Cip/Kip CDK inhibitors display tumor suppressor activity and are frequently inactivated in human tumors. Transcriptional control is also mediated by the Cip/Kip family of CKIs (p21, p27, and p57). p21 directly binds and inhibits E2Fs. p27 localizes to multiple gene promoters with p130-E2F4. Conversely, CKI's may be oncogenic. Indeed, p53-independent p21 induction in human tumors is associated with de-regulated replication factor licencing, and subsequent replication stress.
CDK Inhibitors and Quiescence
Although p21CIP1 and p27Kip1 inhibit the “non G1” cyclin-CDK complexes, they stimulate formation of catalytically active cyclin D-CDK4/6 complexes. During the quiescent G0 state, high levels of p27Kip1 and p21Cip1 bind and inhibit the few cyclin E-CDK2 complexes present. Upon stimulation by extracellular mitogens, cyclin D-CDK6 complexes accumulate in early/mid G1, binding free p21Cip1 and p27Kip1 but retaining catalytic CDK activity. Eventually increased cyclin D-CDK4/6 levels titrate out the Cip/Kip CdkIs, freeing cyclin E-CDK2 complexes which trigger progression through the G1 restriction point.
CDK Knockout mouse models
Knock out mouse models have proven that interphase CDKs can be dispensible for mammalian cell cycle progression, but are required for function of specialized cell types such as hematopoietic cells in Cdk6-/-, endocrine cells in Cdk4-/-, and meiotic germ cells in Cdk2-/-.
In contrast to cell cycle regulation, cyclin and Cdk members involved in transcriptional control are non redundant as ablation results in embryonic lethality. This phenomenon has been observed for Cdk7, Cdk8, Cdk11, cyclin H, cyclin T2, and cyclin K. These cyclin and CDK members regulate transcription through phosphorylation of the RNA polymerase II carboxyl-terminal domain (CTD).
Notably, cyclin D1 appears to directly regulate transcription. Numerous transcriptional regulators/transcription factors have been identified as cyclin D1 binding partners via mass spectrometry, and ChIP-chip identified >900 promoter regions bound by cyclin D1.
CDK inhibitor knockout mouse models
Both Ink4 and Cip/Kip CDK inhibitors display tumor suppressor activity and are frequently inactivated in human tumors. Genetic ablation causes more pronounced overt phenotypes due to compensatory mechanisms masking effects of individual knockouts. For example, p16; p18 KO + p27 deficiency resuts in lymphoma or pituitary tumor development. In fact, in vivo abberation of cell-cycle components most frequently results in pituitary hyperplasias or tumors. Knock-in mice with the CDK4 R24C mutation that prevents effects of the Ink4 CdkIs on Cdk4 results in a wide spectrum of tumors.
Early studies of the root tips of plants identified distinct stages which define the life cycle of a cell. After introduction of radioactive nucleotides at different stages, it was observed that the cells would incorporate the radioactive nucleotides into their DNA only at certain times. Such studies culminated in the definition of cell cycle stages. Cells may exist in a state of quiescence (G0) or may enter the cell cycle. A typical cell has 24 h cell cycle which is generally defined as follows:
Cell Cycle Stages:
- G0 = Quiescence, an exit from cell cycle
- G1 (12-15 h) = Gap Phase 1, when cell is most affected by environmental signals
- S (6-8 h) = DNA Synthesis Phase, when cells incorporate radioactive nucleotides as DNA is replicated
- G2 (2-3 h) = Gap Phase 2, cessation of synthesis before mitosis
- M (~ 1h) = Mitosis, phase in which replicated genome is sequestered to form daughter cell
Cyclin Dependent Kinases:
The core components of the cell cycle regulatory machinery are a family of serine/threonine kinases called the cyclin dependent kinases (CDKs). CDK activity depends on association with regulatory subunits called cyclins which are defined by a conserved 100-amino acid residue-long sequence involved in CDK-binding and activation. While endogenous CDK levels remain relatively consistent, cyclin protein levels fluctuate dramatically in accordance with cell cycle stage. Upon stabilization of a certain cyclin, association with the appropriate CDK forms a cyclin-CDK complex in which the cyclin enhances CDK activity, and directs the complex to the appropriate target protein.
Fluctuation of Cyclin Levels
Fluctuating cyclin levels cause distinct cyclin-CDK complexes to form in a cell cycle-dependent manner. During G1, D-type cyclins (D1, D2, and D3) bind two similarly acting CDKs; CKD4 and CDK6. After the restriction point (R-point) in late G1, E-type cyclins (E1 and E2) associate with CDK2. Cyclin E-CDK2 complexes phosphorylate substrates which promote entry into S-phase, upon which A-type cyclins (A1 and A2) replace E-type cyclins to form cyclin A-CDK2 complexes. As S-phase progresses, the A-type cyclins leave CDK2 to associate with CDC2 (aka CDK1). In late G2 phase, B-type cyclins (B1 and B2) replace A-type cyclins to form Cyclin B- CDC2 complexes which trigger many of the events of mitosis.
Rapid increase of one type of cyclin just prior to cyclin-CDK complex formation occurs in accordance with rapid degradation of the preceding prominent cyclin. This fluctuation occurs through a feedback system where cyclin-CDK complexes in one phase of cell cycle activate those in the subsequent phase, and inactivate those that were active in the previous phase. After the G1 restriction point, cyclin E levels increase. During the G1/S transition, cyclin E-CDK2 complexes activate cyclin A. Cyclin A-CDK2 complexes inactivate the transcription factor which induced cyclin E levels, thus resulting in collapse of cyclin E levels upon S phase entry after cyclin A levels increase. Cyclin A levels decrease upon G2 entry after cyclin A-CDK2 complexes activate cyclin B. Thus, beyond the G1 restriction point, cyclin-CDK complex formation and activation occurs in an autonomous manner through a feedback system independent of extracellular signals. This process ensures completion of cell cycle progression after the commitment is made to cross the G1 restriction point.
Prior to the G1 restriction point, extracellular signals influence the decision for cell cycle entry through control over D-type cyclin levels. Different types of D-type cyclins are activated by distinct signalling pathways which are stimulated by different surface receptors. For example, mitogenic growth factors activate receptor tyrosine kinases on the cell surface to induce signalling cascades which ultimately result in the AP-1 transcription factor binding to and activating the cyclin D1 promoter, resulting in rapid accumulation of cyclin D1. Removal of growth factors from a cell’s medium results in an equally rapid collapse of cyclin D1 levels.
Cyclin Activating Kinases
There are currently 20 members of the Cdk family, each characterized by a conserved catalytic core made of an ATP-binding pocket, a PSTAIRE-like cyclin-binding domain, and an activating T-loop motif.
CDK activation minimally depends on 2 events: 1) Binding to a cyclin; 2) Phosphorylation of a conserved theronine residue on the activating CDK T-loop. In metazoans, CDK 7 appears to be the CDK-activating kinase (CAK) for CDK 4/6, CDK 2, and CDK 1. CDK7 forms a trimeric complex with cyclin H and the RING-finger protein Mat 1. Expression and activity of the 3-subunit CAK complex are constant throughout the cell cycle of actively proliferating cells.
CDK Inhibitors
CDK inhibitors (CdkIs) antagonize the activities of the cyclin-CDK complexes, thus are negative regulators of the cell cycle. These inhibitors are induced by antimitogenic signals. One group of CdkIs called INK4 proteins (originally inhibitors of CDK4) target the G1 cyclin-CDK complexes (CDK4 and CDK6 complexes). The four known INK4 proteins, p16INK4A, p15INK4B, p18INK4C, and p19INK4D, inhibit Cdk4/6 activity by allosteric competitionof their binding sites with cyclins. Mechanistically, INK4A proteins are known to distort both the cyclin-binding site and the ATP-binding site of CDK6 to compromise catalytic activity. The Cip/Kip group of CdkIs inhibit all other cyclin-CDK complexes which form in later stages of cell cycle, and consist of the following members: p21Cip1 (aka p21Waf1), p27Kip1, and p57Kip2. Co-crystallization reveals how one domain of p27Kip1 obstructs the ATP-binding site in the catalytic cleft of CDK2.
Extracellular signals can influence the cell cycle through control over levels and localization of the CDK inhibitors. For example, TGF-β antagonizes cell proliferation through modulation of CdkI levels. TGF-β activation of the TGF-β receptor results in increased levels of p15INK4B which inhibits cyclin D-CDK4/6 complex to prevent cell cycle progression beyond the G1 restriction point. TGF-β also weakly induces p21Cip1 which blocks activity of the remaining “non-G1” cyclin-CDK complexes. More potent induction of p21Cip1 occurs in response to physiological stresses such as DNA damage. Until the genome is repaired, p21Cip1 inhibits cyclin E-CDK2 complexes to block cell cycle advance beyond the G1 restriction point, and inhibits PCNA (proliferating-cell nuclear antigen) which is a key component of the DNA replication machinery.
In contrast to the above examples of extracellular growth-inhibitory signals, growth-promoting mitogens stimulate cell cycle progression though inhibition of CdkIs. For example, mitogens stimulate receptor tyrosine kinases which activate the phosphatidylinositol 3-kinase (PI3K) pathway, leading to activation of the Akt/PKB kinase. Akt/PKB phosphorylates p21Cip1 in the nucleus, inducing nuclear export. Akt/PKB also phosphorylates p27Kip1 to prevent nuclear import.
Both Ink4 and Cip/Kip CDK inhibitors display tumor suppressor activity and are frequently inactivated in human tumors. Transcriptional control is also mediated by the Cip/Kip family of CKIs (p21, p27, and p57). p21 directly binds and inhibits E2Fs. p27 localizes to multiple gene promoters with p130-E2F4. Conversely, CKI's may be oncogenic. Indeed, p53-independent p21 induction in human tumors is associated with de-regulated replication factor licencing, and subsequent replication stress.
CDK Inhibitors and Quiescence
Although p21CIP1 and p27Kip1 inhibit the “non G1” cyclin-CDK complexes, they stimulate formation of catalytically active cyclin D-CDK4/6 complexes. During the quiescent G0 state, high levels of p27Kip1 and p21Cip1 bind and inhibit the few cyclin E-CDK2 complexes present. Upon stimulation by extracellular mitogens, cyclin D-CDK6 complexes accumulate in early/mid G1, binding free p21Cip1 and p27Kip1 but retaining catalytic CDK activity. Eventually increased cyclin D-CDK4/6 levels titrate out the Cip/Kip CdkIs, freeing cyclin E-CDK2 complexes which trigger progression through the G1 restriction point.
CDK Knockout mouse models
Knock out mouse models have proven that interphase CDKs can be dispensible for mammalian cell cycle progression, but are required for function of specialized cell types such as hematopoietic cells in Cdk6-/-, endocrine cells in Cdk4-/-, and meiotic germ cells in Cdk2-/-.
In contrast to cell cycle regulation, cyclin and Cdk members involved in transcriptional control are non redundant as ablation results in embryonic lethality. This phenomenon has been observed for Cdk7, Cdk8, Cdk11, cyclin H, cyclin T2, and cyclin K. These cyclin and CDK members regulate transcription through phosphorylation of the RNA polymerase II carboxyl-terminal domain (CTD).
Notably, cyclin D1 appears to directly regulate transcription. Numerous transcriptional regulators/transcription factors have been identified as cyclin D1 binding partners via mass spectrometry, and ChIP-chip identified >900 promoter regions bound by cyclin D1.
CDK inhibitor knockout mouse models
Both Ink4 and Cip/Kip CDK inhibitors display tumor suppressor activity and are frequently inactivated in human tumors. Genetic ablation causes more pronounced overt phenotypes due to compensatory mechanisms masking effects of individual knockouts. For example, p16; p18 KO + p27 deficiency resuts in lymphoma or pituitary tumor development. In fact, in vivo abberation of cell-cycle components most frequently results in pituitary hyperplasias or tumors. Knock-in mice with the CDK4 R24C mutation that prevents effects of the Ink4 CdkIs on Cdk4 results in a wide spectrum of tumors.