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Interaction of CDK inhibitors with PCNA The mammalian protein p21, induced by the tumour-suppressor protein p53, interacts
with and inhibits two different targets essential for cell-cycle progression. One of these is the
cyclin-Cdk family of kinases and the other is the essential DNA replication factor,
proliferating-cell nuclear antigen (Drosophila homolog: PCNA). Separate domains of p21 are
responsible for interacting with and inhibiting the two targets. An amino-terminal domain inhibits
cyclin-Cdk kinases and a carboxy-terminal domain inhibits PCNA. p21 inhibits different biological systems through different
targets. The PCNA-binding domain is sufficient for inhibition of DNA replication based on
simian virus 40, whereas the Cdk2-binding domain is sufficient for inhibition of DNA replication
based on Xenopus egg extract and for growth suppression in transformed human cells (J. Chen, 1995).
Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, it has been demonstrated
that the cyclin E protein, in association with Cdk2, is required for the elongation phase of DNA
replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target
by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded
replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex
appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin
A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The
provision of a large maternal pool of cyclin E protein shows that regulators of replication are
constitutively present, which explains the lack of a protein synthesis requirement for replication
in the early embryonic cell cycle (Jackson, 1995).
DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated
mammalian DNA, but the factors regulating this activity are unknown. MCMT
is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for
DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of
MCMT; it occurs in intact cells at foci of newly replicated DNA and does not alter
MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can
disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate
methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be
linked in a regulatory pathway, because the extents of their expression are inversely
related in both SV40-transformed and nontransformed cells (Chuang, 1997).
Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK)
inhibitors. The p21 family currently includes three distinct genes, p21, p27(Kip1), and p57(Kip2), that
share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin
complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate
C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain
unique C-terminal domains whose functions are unknown. The human p57 protein,
like p21, contains a PCNA-binding domain within its C terminus. When its
N-terminal CDK-cyclin binding domain is removed, the altered p57 can prevent DNA replication in vitro and S phase entry in
vivo. Disruption of either CDK/cyclin or PCNA binding partially reduces p57's ability to suppress
myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely
eliminates p57's suppressive activity. Thus, control of cell cycle and suppression of cell transformation
by p57 requires both CDK and PCNA inhibitory activity, and disruption of either or both functions may
lead to uncontrolled cell growth (Watanabe, 1998).
The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, a new assay was devised for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. RFC is shown to remain transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 does not inhibit RFC-dependent PCNA loading; rather, p21 forms a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA results in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilizes the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex is no longer displaced from the DNA by p21. It is proposed that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. It is also suggested that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome (Waga, 1998).
One amino-terminal p21 peptide spanning amino acids
15-40 antagonizes p21 binding and inhibition of cyclin E/Cdk2 kinase. However, antagonism of p21
binding is lost in a similar peptide lacking amino acids 15-20, as well as in a peptide in
which cysteine-18 is substituted for a serine. These results suggest that this peptide region is
important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also
antagonizes p21-activity, but this peptide does not affect the ability of p21 to interact with cyclin
E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition
because none of the peptides tested inhibits cyclin E/Cdk2. A peptide
spanning amino acids 21-45 binds recombinant p21: for this interaction, amino acids 26 through 45 are required. A p21 peptide
spanning amino acids 139-164 binds PCNA in a filter binding assay: this peptide
suppresses recombinant p21-PCNA interaction. Conformational analysis reveals that peptides
spanning amino acids 21-45 and 139-164 tend towards an alpha-helical conformation, indicating that these regions are probably in a coiled conformation in the
native protein. Taken together, these results provide an insight into domains of p21 that are
involved in cyclin E/Cdk2 and PCNA interaction and also suggest that a potential p21
dimerization domain may lie in the amino-terminus of p21 (I. Chen, 1995).
The DNA polymerase delta processivity factor PCNA promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4Cdt2 E3 ubiquitin ligase complex. PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4Cdt2 ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4Cdt2 E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, this study shows that the CRL4Cdt2 and the SCFSkp2 ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle (Abbas, 2008).
Interaction of CDK inhibitors with RB family proteins The Retinoblastoma-related protein p107 (See Drosophila Retinoblastoma-family protein), like the p21 family of CDK inhibitors, can inhibit the phosphorylation of target substrates by cyclin A/cdk2 and cyclin E/cdk2 complexes. The associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Interactions between p107 and p21 are mutually exclusive: p21 causes a dissociation of p107/cyclin/cdk2 complexes yielding in their place, p21/cyclin/cdk2 complexes. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. It has been suggested that p107 functions in a manner similar to Rb, causing growth arrest of sensitive cells in the G1 phase of the cell cycle. The p107 molecule can be dissected into two domains, either of which is able to independently block cell cycle progression. One domain corresponds to the sequences needed for interaction with transcription E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes (Zhu, 1995 a and b).
The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction
point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has
also been shown to monitor progression of G1 phase, mostly through the interaction with E2F family members. p107 is able
to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. pRb2/p130 also possesses this activity, but through a separate domain. The increased
expression of pRb2/p130 during various cellular processes is correlated with the decreased kinase activity of Cdk2. It is hypothesized that
pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a
manner different from p107 (De Luca, 1997).
Mice lacking functional Rb die at midgestation and
exhibit ectopic DNA synthesis, apoptosis, and incomplete
differentiation during neurogenesis, erythropoiesis, and
lens development. Temporal and genetic analyses are described for partially rescued Rb mutant fetuses, mgRb:Rb-/-, that survive to birth. Specific defects in skeletal muscle differentiation are revealed. Increased apoptosis, bona fide endoreduplication, and incomplete differentiation throughout terminal myogenesis exhibited by mgRb:Rb-/- fetuses is further augmented in composite mutant fetuses, mgRb:Rb-/-:p21-/-, lacking both Rb and the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Although E2F1 and p53 mediate ectopic DNA synthesis and cell death in several tissues in Rb mutant embryos, both endoreduplication and apoptosis persist in mgRb:Rb-/-:E2F1-/- and mgRb:Rb-/-:p53-/- compound mutant muscles. Thus, combined inactivation of Rb and p21Waf1/Cip1 augments endoreduplication and apoptosis, whereas
E2F1 and p53 are dispensable during aberrant myogenesis in Rb-deficient fetuses.
These results show that ectopic DNA synthesis and apoptosis in
Rb-deficient muscles are mediated by a pathway, yet to be defined, which is independent of E2F1 or p53. Inactivation of both Rb and p21Waf1/Cip1 leads to increased endoreduplication and apoptosis, indicating that these two negative regulators cooperate to facilitate cell cycle exit during terminal myogenesis (Jiang, 2000).
The tumor suppressor p27Kip1 is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27Kip1 also has cell cycle-independent functions. Indeed, p27Kip1 regulates cell migration, as evidenced by the observation that p27Kip1-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27Kip1 is independent of its cell-cycle regulatory functions; re-expression of both wild-type p27Kip1 and a mutant p27Kip1 (p27CK–) that cannot bind to cyclins and CDKs rescues migration of p27–/– cells. p27–/– cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27Kip1. Moreover, inhibition of ROCK, a downstream effector of Rho, is able to rescue the migration defect of p27–/– cells in response to growth factors. Finally, p27Kip1 is found to bind to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine–nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27Kip1 in regulating cell migration via modulation of the Rho pathway (Besson, 2004).
It appears that all of the members of the Cip/Kip family of CKIs may play a role in the regulation of the Rho pathway, albeit acting at distinct levels in the pathway. p27Kip1 has been shown in this study to regulate Rho activation. Cytoplasmic p21Waf1/Cip1 has been shown to directly inhibit ROCK, resulting in increased neurite outgrowth (Tanaka, 2002; Lee, 2003). However, in the current experiments the loss of p21Waf1 in MEFs had no effect on their migratory ability. p57Kip2 binds to LIM-kinase and induces its translocation to the nucleus, thereby inhibiting its activity (Yokoo, 2003). LIM-kinase phosphorylates and inactivates the actin depolymerization factor cofilin, and is itself directly activated by ROCK phosphorylation. Therefore, although they act differently, all of the Cip/Kip proteins seem to negatively regulate the Rho signaling pathway when in the cytoplasm. It is tempting to speculate that the regulation of different proteins in the Rho pathway by CKIs could provide new levels of regulation of Rho-mediated processes, because the abundance and subcellular localization of CKIs are regulated throughout the cell cycle (Besson, 2004).
Genome stability requires that genomic DNA is replicated only once per cell cycle. The replication-licensing system ensures that the formation of prereplicative complexes is temporally separated from the initiation of DNA replication. The replication-licensing factors Cdc6 and Cdt1 are required for the assembly of prereplicative complexes during G1 phase. During S phase, metazoan Cdt1 is targeted for degradation by the CUL4 ubiquitin ligase, and vertebrate Cdc6 is translocated from the nucleus to the cytoplasm. However, because residual vertebrate Cdc6 remains in the nucleus throughout S phase, it has been unclear whether Cdc6 translocation to the cytoplasm prevents rereplication. The inactivation of C. elegans CUL-4 is associated with dramatic levels of DNA rereplication. This study shows that C. elegans CDC-6 is exported from the nucleus during S phase in response to the phosphorylation of multiple CDK sites. CUL-4 promotes the phosphorylation and subsequent translocation of CDC-6 via negative regulation of the CDK-inhibitor CKI-1. Rereplication can be induced by coexpression of nonexportable CDC-6 with nondegradable CDT-1, indicating that redundant regulation of CDC-6 and CDT-1 prevents rereplication. This demonstrates that CDC-6 translocation is critical for preventing rereplication and that CUL-4 independently controls both replication-licensing factors (Kim, 2007).
In humans and Xenopus, ectopically expressed Cdc6 is completely exported from the nucleus during S phase; in contrast, a substantial fraction of endogenous Cdc6 remains nuclear localized during S phase. Strikingly, a similar result is observed in C. elegans, with a substantial fraction of endogenous CDC-6 remaining in the nucleus during S phase, whereas ectopically expressed CDC-6 appears exclusively cytoplasmic. The reason(s) for these differential localizations are not understood (Kim, 2007).
The presence of nuclear-localized Cdc6 during S phase in mammalian cells has led to the proposal that Cdc6 translocation is not important for restraining DNA-replication licensing. Further, there is currently no evidence for a functional role of Cdc6 translocation in preventing rereplication. In this study, it was observed that nonexportable CDC-6 can synergize with deregulated CDT-1 to induce rereplication. This implies that CDC-6 translocation is a redundant safeguard to prevent the reinitiation of DNA replication. This provides the first evidence in any organism of a functional role for phosphorylation-dependent CDC-6 nuclear export (Kim, 2007).
In S. pombe, the overexpression of the Cdc6 ortholog (Cdc18) is sufficient to induce significant rereplication. In contrast, overexpression of Cdc6 does not induce rereplication in S. cerevisiae, Drosophila, or humans. In humans, co-overexpression of wild-type Cdt1 and Cdc6 in cells that lack a cell-cycle checkpoint produces only modest rereplication in a subset of cells (Kim, 2007).
Coexpression of nondegradable CDT-1 and nonexportable CDC-6 produces significant rereplication in a subset of early-stage C. elegans embryos. In contrast, overexpression of combinations of deregulated and wild-type CDT-1 or CDC-6 does not induce rereplication. This indicates that redundant regulation of CDT-1 and CDC-6 prevents rereplication. No rereplication was observed in every embryonic cell expressing deregulated CDT-1 and CDC-6. This suggests that there might be additional mechanisms that act in the early embryo to limit rereplication (Kim, 2007).
The expression of combinations of wild-type and deregulated CDT-1 and CDC-6 produced an embryonic lethality that was not associated with increased DNA levels. The cause of this lethality is unclear, but it might arise from changes in DNA-replication timing, which is known to produce embryonic arrest (Kim, 2007).
Inactivation of CUL-4 produces dramatic levels of rereplication that are associated with a failure to degrade CDT-1. However, overexpressing Cdt1 in fission yeast does not induce rereplication, and overexpressing human Cdt1 several-log-fold higher than the endogenous protein produces only modest rereplication in a subset of cells. Given the negligible or limited effects of greatly overexpressing Cdt1 in other organisms, it was hard to reconcile the substantial rereplication associated with merely failing to degrade CDT-1 during S phase in cul-4(RNAi) animals (Kim, 2007).
This work reveals that the CDC-6 replication-licensing factor is also deregulated in cul-4(RNAi) animals. CDC-6 remains nuclear throughout S phase in cul-4(RNAi) animals, and this is correlated with a failure to phosphorylate CDC-6 on CDK sites. CUL-4 negatively regulates the levels of the CDK inhibitor CKI-1. The negative regulation of CKIs of the CIP/KIP family by CUL-4 is conserved in Drosophila and humans. cki-1 RNAi suppresses rereplication in cul-4 mutants without affecting CDT-1 accumulation, indicating that CKI-1 is independently required for the induction of rereplication. Significantly, the presence of CKI-1 is required for the block on CDC-6 phosphorylation and nuclear export in cul-4(gk434) cells. These results suggest that CUL-4 promotes CDC-6 nuclear export by negatively regulating CKI-1 levels, thereby allowing CDK(s) to phosphorylate CDC-6 and induce its nuclear export. The evidence that CDK(s) are the relevant kinases is that CDC-6 is phosphorylated on CDK consensus sites and the phosphorylation is blocked by a CDK inhibitor. In yeast and mammals, CDK activity prevents rereplication, and siRNA codepletion of CDK1 and CDK2 in human cells induces limited rereplication. These results suggest that in metazoa, Cdc6 is one of the critical targets of CDKs for preventing rereplication. This work further indicates that CUL-4 is a master regulator that restrains DNA replication through two independent pathways: mediating CDT-1 degradation and promoting CDC-6 nuclear export via the negative regulation of CKI-1 (Kim, 2007).
Other interactions of CDK inhibitors p21WAF1/CIP1, which belongs to a class of regulatory proteins that interact with cyclin dependent kinases is a potent inhibitor of these kinases. The inhibition of the cyclin dependent kinases induces an arrest of cells in the G phase of the cell cycle. In addition p21WAF1/CIP1 associates with PCNA and inhibits DNA replication. p21WAF1/CIP1 binds to the regulatory beta-subunit of protein kinase CK2 (See Drosophila Casein kinase II) but not to the catalytic alpha-subunit. Binding of p21WAF1/CIP1 down regulates the kinase activity of CK2 with respect to the phosphorylation of the beta-subunit of CK2, casein and the C-terminus of p53. This study demonstrates a new binding partner for the regulatory beta-subunit of protein kinase CK2 which regulates the activity of the holoenzyme (Gotz, 1996).
CCAAT/enhancer binding protein alpha (C/EBP alpha) (Drosophila homolog: Slow border cells) is expressed at high levels in quiescent
hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in
part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation
in vivo is presented in this paper. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in
the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the
hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life, when
cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type
neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly
proliferating livers, which lack C/EBP alpha, suggests that p21 is responsible for C/EBP alpha-mediated
control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP
alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery
levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by
C/EBP alpha in liver. Coimmunoprecipitation and a mammalian two-hybrid assay system reveals the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear
extracts showsthat C/EBP alpha blocks proteolytic degradation of p21. These data demonstrate that
C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21
protein is under posttranscriptional control, consistent with the hypothesis that protein-protein
interaction with C/EBP alpha determines p21 levels (Timchenko, 1997).
The regulation of the c-Jun NH2-terminal kinase (JNK) subfamily of mitogen-activated protein kinases
(MAPKs), in response to the inhibition of DNA replication, was examined during the cell cycle of human T-lymphocytes. JNK is rapidly activated following release of T-lymphocytes from G1/S-phase arrest and this activation
precedes resumption of DNA synthesis upon S-phase progression. Activation of JNK correlates with
dissociation of the cyclin-dependent protein kinase (CDK) inhibitor, p21WAF1, from JNK1. Since JNK1 isolated from
T-lymphocytes by immunoprecipitation can be inhibited by recombinant p21WAF1 in vitro, these data suggest that JNK
activation may be regulated in part by its dissociation from p21WAF1. The observation of a dynamic, physical association of
native JNK1 and p21WAF1 in vivo has not previously been described and suggests a novel mechanism for JNK-mediated
regulation of the cell cycle of human T-lymphocytes (Patel, 1998).
Fibroblast growth factors (FGFs) are upstream activators of the MAP kinase pathway and mitogens in a wide variety of cells. However, whether the MAP kinase pathway solely accounts for the induction of cell cycle or anti-apoptotic activity of the FGF receptor (FGFR) tyrosine kinase is not clear. Cell cycle inducer Cks1 (See Drosophila Cyclin-dependent kinase subunit) that triggers ubiquitination and degradation of p27kip1 associates with the unphosphorylated form of FGFR substrate 2 (FRS2), an adaptor protein that is phosphorylated by FGFR kinases and recruits downstream signaling molecules. FGF-dependent activation of FGFR tyrosine kinases induces FRS2 phosphorylation, causes release of Cks1 from FRS2, and promotes degradation of p27kip1 in 3T3 cells. Since degradation of p27kip1 is a key regulatory step in activation of the cyclin E/A-Cdk complex during the G1/S transition of the cell cycle, the results suggest a novel mitogenic pathway whereby FGF and other growth factors that activate FRS2 directly activate cyclin-dependent kinases (Zhang, 2004).
Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. SCML2B was purified and found to form a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals (Leconam 2013).
CDK inhibitors as targets of the Ras pathway While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenically transformed
cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage
dependence is not fully understood. When normal rat fibroblasts are cultured in suspension without substrate attachment, the
cell cycle arrests in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulate.
Conditional expression of oncogenic Ras (Drosophila homolog: Ras85D) induces the G1-S transition of the cell cycle and significantly shortens the half-life of
p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase (Drosophila homolog: Rolled) by
cyclic AMP-elevating agents and a MEK inhibitor prevents the oncogenic Ras-induced degradation of p27Kip1. These
results suggest that the loss of substrate attachment induces cell cycle arrest through the up-regulation of p27Kip1 mRNA,
but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the
MAP kinase signaling pathway. It appears that p27Kip1 is phosphorylated by MAP kinase in vitro and the
phosphorylated p27Kip1 cannot bind to and inhibit cdk2 (Kawada, 1997).
Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of
GTPases, one of their most important roles is as regulators of cell proliferation: the mitogenic response to a variety of
growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signaling
pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular
responses by activating Ras-dependent signaling pathways to varying degrees. Other signaling pathways could also influence
the consequences of Ras signaling. When signaling through the Ras-related GTPase Rho is inhibited,
constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase
of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA
synthesis. Therefore, Rho helps Ras to drive cells into S phase. Cells that lack p21Waf1/Cip1 do not require Rho signaling for the induction of DNA synthesis by activated Ras,
indicating that, once Ras has become activated, the primary requirement for Rho signaling is the suppression of p21Waf1/Cip1 induction (Olson, 1998).
Cdk inhibitors and G2 arrest Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by
inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1).
In addition to exerting negative control of the G1/S phase transition, p21 may
play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the
nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and
associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear
accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing
the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in
tumor-derived cells. Synchronized E6-expressing fibroblasts show accelerated entry into
mitosis, when compared to wild-type cells, and exhibit higher cyclin A- and cyclin B1-associated kinase
activities. Primary embryonic fibroblasts derived from p21-/- mice have significantly reduced
numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient
pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls (Dulic, 1998).
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