The restriction point (R), also known as the Start or G
1/S checkpoint, is a
cell cycle checkpoint
Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, wi ...
in the
G1 phase of the animal
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
at which the cell becomes "committed" to the cell cycle, and after which extracellular
signals
In signal processing, a signal is a function that conveys information about a phenomenon. Any quantity that can vary over space or time can be used as a signal to share messages between observers. The ''IEEE Transactions on Signal Processing'' ...
are no longer required to stimulate proliferation.
The defining biochemical feature of the restriction point is the activation of G
1/S- and
S-phase
S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during ...
cyclin-CDK complexes, which in turn
phosphorylate
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, ...
proteins that initiate
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
,
centrosome
In cell biology, the centrosome (Latin centrum 'center' + Greek sōma 'body') (archaically cytocentre) is an organelle that serves as the main microtubule organizing center (MTOC) of the animal cell, as well as a regulator of cell-cycle prog ...
duplication, and other early cell cycle events. It is one of three main cell cycle checkpoints, the other two being the
G2-M DNA damage checkpoint
The G2-M DNA damage checkpoint is an important cell cycle checkpoint in eukaryotic organisms that ensures that cells don't initiate mitosis until damaged or incompletely replicated DNA is sufficiently repaired. Cells with a defective G2-M checkpo ...
and the
spindle checkpoint
The spindle checkpoint, also known as the metaphase-to-anaphase transition, the spindle assembly checkpoint (SAC), the metaphase checkpoint, or the mitotic checkpoint, is a cell cycle checkpoint during mitosis or meiosis that prevents the separa ...
.
History
Originally,
Howard Martin Temin
Howard Martin Temin (December 10, 1934 – February 9, 1994) was an American geneticist and virologist. He discovered reverse transcriptase in the 1970s at the University of Wisconsin–Madison, for which he shared the 1975 Nobel Prize in Phys ...
showed that chicken cells reach a point at which they are committed to replicate their DNA and are not dependent on extracellular signals.
About 20 years later, in 1973,
Arthur Pardee
Arthur Beck Pardee (July 13, 1921 – February 24, 2019) was an American biochemist. One biographical portrait begins "Among the titans of science, Arthur Pardee is especially intriguing." There is hardly a field of molecular biology that is not ...
demonstrated that a single restriction point exists in
G1. Previously, G
1 had been defined simply as the time between mitosis and
S phase. No molecular or morphological place-markers for a cell's position in G
1 were known. Pardee used a double-block method in which he shifted cells from one cell cycle block (such as critical amino acid withdrawal or serum withdrawal) to another and compared each block's efficiency at preventing progression to S phase. He found that both blocks in all cases examined were equally efficient at blocking S phase progression, indicating that they must all act at the same point in G
1, which he termed the "restriction point", or R-point.
In 1985, Zetterberg and Larsson discovered that, in all stages of the cell cycle, serum deprivation results in inhibition of protein synthesis. Only in postmitotic cells (i.e. cells in early G
1) did serum withdrawal force cells into quiescence (
G0). In fact, Zetterberg found that virtually all of the variability in cell cycle length can be accounted for in the time it takes the cell to move from the restriction point to S phase.
Extracellular signals
Except for early embryonic development, most cells in multicellular organisms persist in a quiescent state known as G
0, where proliferation does not occur, and cells are typically terminally differentiated; other specialized cells continue to divide into adulthood. For both of these groups of cells, a decision has been made to either exit the cell cycle and become quiescent (G
0), or to reenter G
1.
A cell's decision to enter, or reenter, the cell cycle is made before S-phase in G
1 at what is known as the restriction point, and is determined by the combination of promotional and inhibitory extracellular signals that are received and processed. Before the R-point, a cell requires these extracellular stimulants to begin progressing through the first three sub-phases of G
1 (competence, entry G
1a, progression G
1b). After the R-point has been passed in G
1b, however, extracellular signals are no longer required, and the cell is irreversibly committed to preparing for
DNA duplication. Further progression is regulated by intracellular mechanisms. Removal of stimulants before the cell reaches the R-point may result in the cell's reversion to quiescence.
Under these conditions, cells are actually set back in the cell cycle, and will require additional time (about 8 hours more than the withdrawal time in culture) after passing the restriction point to enter S phase.
Mitogen Signaling
Growth factors (e.g.,
PDGF, FGF, and
EGF) regulate entry of cells into the cell cycle and progression to the restriction point. After passing this switch-like “point of no return,” cell cycle completion is no longer dependent on the presence of mitogens.
Sustained mitogen signaling promotes cell cycle entry largely through regulation of the G1 cyclins (cyclin D1-3) and their assembly with Cdk4/6, which may be mediated in parallel through both MAPK and PI3K pathways.
''MAPK Signaling Cascade''
The binding of extracellular growth factors to their
receptor tyrosine kinase
Receptor tyrosine kinases (RTKs) are the high- affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Of the 90 unique tyrosine kinase genes identified in the human genome, 58 encode receptor tyrosine kin ...
s (RTK) triggers a conformational change and promotes dimerization and autophosphorylation of tyrosine residues on the cytoplasmic tail of the RTKs. These phosphorylated tyrosine residues facilitate the docking of proteins containing an SH2-domain (e.g.,
Grb2), which can subsequently recruit other signaling proteins to the plasma membrane and trigger signaling kinase cascades. RTK-associated Grb2 binds
Sos, which is a guanine nucleotide exchange factor that converts membrane-bound
Ras to its active form (Ras-GDP
Ras-GTP).
Active Ras activates the MAP kinase cascade, binding and activating Raf, which phosphorylates and activates MEK, which phosphorylates and activates
ERK (also known as MAPK, ''see also
MAPK/ERK pathway'').
Active ERK then translocates into the nucleus where it activates multiple targets, such as the transcription factor serum-response factor (SRF), resulting in expression of immediate early genes—notably the transcription factors
Fos and
Myc
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' (MYC), ''l-myc'' ( MYCL), and ''n-myc'' (MYCN). ''c-myc'' (also sometimes refe ...
.
Fos/Jun dimers comprise the transcription factor complex
AP-1 and activate delayed response genes, including the major
G1 cyclin,
cyclin D1.
Myc also regulates expression of a wide variety of pro-proliferative and pro-growth genes, including some induction of cyclin D2 and
Cdk4.
Additionally, sustained ERK activity seems to be important for phosphorylation and nuclear localization of
CDK2
Cyclin-dependent kinase 2, also known as cell division protein kinase 2, or Cdk2, is an enzyme that in humans is encoded by the ''CDK2'' gene. The protein encoded by this gene is a member of the cyclin-dependent kinase family of Ser/Thr protein ...
,
further supporting progression through the restriction point.
''PI3K Pathway Signaling''
p85, another SH2-domain-containing protein, binds activated RTKs and recruits
PI3K
Phosphoinositide 3-kinases (PI3Ks), also called phosphatidylinositol 3-kinases, are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which i ...
(phosphoinositide-3-kinase), phosphorylating the phospholipid PIP2 to PIP3, leading to recruitment of
Akt
Protein kinase B (PKB), also known as Akt, is the collective name of a set of three serine/threonine-specific protein kinases that play key roles in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, tran ...
(via its PH-domain). In addition to other pro-growth and pro-survival functions, Akt inhibits glycogen synthase kinase-3β (
GSK3β
Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased suscept ...
), thereby preventing GSK3β -mediated phosphorylation and subsequent degradation of cyclin D1 (''see figure''). Akt further regulates G1/S components by mTOR-mediated promotion of cyclin D1 translation, phosphorylation of the Cdk inhibitors
p27kip1 (preventing its nuclear import) and
p21Cip1 (decreasing stability), and inactivating phosphorylation of the transcription factor
FOXO4
Forkhead box protein O4 is a protein that in humans is encoded by the ''FOXO4'' gene.
Structure and function
FOXO4 is a member of the forkhead family transcription factors O subclass, which is characterized by a winged helix domain used for DN ...
(which regulates p27 expression). Together, this stabilization of cyclin D1 and destabilization of Cdk inhibitors favors G1 and G1/S-Cdk activity.
Anti-mitogen Signaling
Anti-mitogens like the cytokine
TGF-β
Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-β 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other sign ...
inhibit progression through the restriction point, causing a G1 arrest. TGF-β signaling activates Smads, which complex with
E2F4
Transcription factor E2F4 is a protein that in humans is encoded by the ''E2F4'' gene.
Function
The protein encoded by this gene is a member of the E2F family of transcription factors. The E2F family plays a crucial role in the control of c ...
/5 to repress Myc expression and also associate with Miz1 to activate expression of the Cdk inhibitor p15
INK4b to block cyclin D-Cdk complex formation and activity.
Cells arrested with TGF-β also accumulate p21 and p27.
Mechanism
''Overview''
As described above, signals from extracellular growth factors are
transduced in a typical manner.
Growth factor
A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regul ...
binds to receptors on the cell surface, and a variety of phosphorylation cascades result in Ca
2+ uptake and protein phosphorylation. Phosphoprotein levels are counterbalanced by phosphatases. Ultimately, transcriptional activation of certain target genes occurs. Extracellular signaling must be maintained, and the cell must also have access to sufficient nutrient supplies to support rapid protein synthesis. Accumulation of
cyclin D's are essential.
Cyclin D-bound
cdks 4 and
6 are activated by
cdk-activating kinase and drive the cell towards the restriction point. Cyclin D, however has a high turnover rate (t
1/2<25 min). It is because of this quick turnover rate that the cell is extremely sensitive to mitogenic signaling levels, which not only stimulate cyclin D production, but also help to stabilize cyclin D within the cell.
In this way, cyclin D acts as a mitogenic signal sensor.
Cdk inhibitors (CKI), such as the
Ink4 proteins and
p21, help to prevent improper cyclin-cdk activity.
Active cyclin D-cdk complexes phosphorylate
retinoblastoma protein
The retinoblastoma protein (protein name abbreviated pRb; gene name abbreviated ''Rb'', ''RB'' or ''RB1'') is a proto-oncogenic tumor suppressor protein that is dysfunctional in several major cancers. One function of pRb is to prevent excessive ...
(pRb) in the nucleus. Unphosphorylated Rb acts as an inhibitor of G
1 by preventing
E2F-mediated transcription. Once phosphorylated, E2F activates the transcription of cyclins E and A.
Active cyclin E-cdk begins to accumulate and completes pRb phosphorylation, as shown in the figure.
''Cdk inhibitors and regulation of Cyclin D/Cdk complex activity''
p27 and p21 are stoichiometric inhibitors of G1/S- and S-cyclin-Cdk complexes. While p21 levels increase during cell-cycle entry, p27 is generally inactivated as cells progress to late G1.
High cell density, mitogen starvation, and TGF-β result in accumulation of p27 and cell cycle arrest.
Similarly, DNA damage and other stressors increase p21 levels, while mitogen-stimulated ERK2 and Akt activity leads to inactivating phosphorylation of p21.
Early work on p27 overexpression suggested that it can associate with and inhibit cyclin D-Cdk4/6 complexes and cyclin E/A-Cdk2 complexes ''in vitro'' and in select cell types.
However, kinetic studies by LaBaer et al. (1997) found that titrating in p21 and p27 promotes assembly of the cyclin d-Cdk complex, increasing overall activity and nuclear localization of the complex. Subsequent studies elucidated that p27 may be required for cyclin D-Cdk complex formation, as p27
-/-, p21
-/- MEFs showed a decrease in cyclin D-Cdk4 complexation that could be rescued with p27 re-expression.
Work by James et al. (2008) further suggests that phosphorylation of tyrosine residues on p27 can switch p27 between an inhibitory and non-inhibitory state while bound to cyclin D-Cdk4/6, offering a model for how p27 is capable of regulating both cyclin-Cdk complex assembly and activity. Association of p27 with cyclin D-Cdk4/6 may further promote cell cycle progression by limiting the pool of p27 available for inactivating cyclin E-Cdk2 complexes.
Increasing cyclin E-Cdk2 activity in late G1 (and cyclin A-Cdk2 in early S) leads to p21/p27 phosphorylation that promotes their nuclear export,
ubiquitin
Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 1980s. Fo ...
ation, and degradation.
Dynamics
A paper published by the Lingchong You and Joe Nevins groups at
Duke University in 2008 demonstrated that the a bistable
hysteric E2F switch underlies the restriction point.
E2F promotes its own activation, and also promotes the inhibition of its own inhibitor (
pRb), forming two
feedback loops (among others) that are important in establishing bistable systems. The authors of this study used a destabilized
GFP-system under the control of the
E2F promoter as a
readout of
E2F activity. Serum-starved cells were stimulated with varying serum concentrations, and the GFP readout was recorded at a single-cell level. They found that the GFP
reporter was either on or off, indicating that
E2F was either completely activated or deactivated at all of the different serum levels analyzed. Further experiments, in which they analyzed the history-dependence of the E2F system confirmed that it operates as a
hysteretic
Hysteresis is the dependence of the state of a system on its history. For example, a magnet may have more than one possible magnetic moment in a given magnetic field, depending on how the field changed in the past. Plots of a single component of ...
bistable switch.
In cancer
Cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
can be seen as a disruption of normal restriction point function, as cells continually and inappropriately reenter the
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
, and do not enter G
0.
Mutations at many steps in the pathway towards the restriction point can result in cancerous growth of cells. Some of the genes most commonly mutated in cancer include Cdks and CKIs; overactive Cdks or underactive CKIs lower the stringency of the restriction point, allowing more cells to bypass senescence.
The restriction point is an important consideration in the development of new drug therapies. Under normal physiological conditions, all cell proliferation is regulated by the restriction point. This can be exploited and used as a way to protect non-cancerous cells from
chemotherapy
Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs ( chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemothe ...
treatments. Chemotherapy drugs typically attack cells that are proliferating rapidly. By using drugs that inhibit completion of the restriction point, such as
growth factor receptor inhibitors, normal cells are prevented from proliferating, and are thus protected from chemotherapy treatments.
See also
*
S-phase-promoting factor
*
Cyclin D
Cyclin D is a member of the cyclin protein family that is involved in regulating cell cycle progression. The synthesis of cyclin D is initiated during G1 and drives the G1/S phase transition. Cyclin D protein is anywhere from 155 (in zebra mu ...
*
MAPK/ERK pathway
*
p21
*
p27
References
{{Cell cycle
Cell cycle