dacapo
Interaction of Dacapo homologs with the cyclin/cdk complex
The D-type cyclins and their major kinase partners CDK4 (Drosophila homolog: Cyclin-dependent kinase 4/6) and CDK6 regulate G0-G1-S progression by
contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of
active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family
members. Although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only
p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other
INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4.
Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes,
suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In
agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to
p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest
that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by
counteracting the effects of INK4 molecules (Parry, 1999).
Specific cyclin-dependent kinases are found to regulate transcriptional activation by NF-kappaB transcription factor through interactions with the transcriptional coactivator p300. The transcriptional activation domain of RelA(p65), a subunit of NF-kappaB (Drosophila homolog Dorsal), interacts with an amino-terminal region of p300 that is distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex (See Drosophila cyclin E). The CDK inhibitor p21 or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates NF-kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21, a Dacapo homolog. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins, 1997).
The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic
activity. p27(Kip1) is regulated at various levels, including translation, degradation by the
ubiquitin/proteasome pathway and non-covalent sequestration. Point mutants of p27 are described that are
deficient in their interaction with either cyclins [p27(c-)], CDKs [p27(k-)] or both [p27(ck-)]. Each contact is critical for kinase inhibition and induction of G1 arrest. Through its
intact cyclin contact, p27(k-) associates with active cyclin E-CDK2 and, unlike wild type p27, p27(c-)
or p27(ck-), is efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site
(TPKK). Retrovirally expressed p27(k-) is rapidly degraded through the proteasome in Rat1 cells,
but is stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting,
exogenous wild-type p27 forms inactive ternary complexes with cellular cyclin E-CDK2, is not
degraded through the proteasome, and is not further stabilized by the VPKK mutation. p27(ck-),
which is not recruited to cyclin E-CDK2, also remains stable in vivo. Thus, selective degradation of
p27(k-) depends upon association with active cyclin E-CDK2 and subsequent phosphorylation.
Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to
be degraded by the proteasome. It is proposed that cellular p27 must also exist transiently in a
cyclin-bound non-inhibitory conformation in vivo (Vlach, 1997).
Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the
cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E
for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as
monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation
by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. Cyclin
E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F
binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein
interaction. These observations suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late
G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant
of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene
requires an active cdk2 kinase tethered to its promoter region (Zerfass-Thome, 1997).
In dividing cells, p27Kip1 is predominantly bound to cyclin D-cdk4 without inhibiting
this kinase. Once induced by TGF-ß or with a conditional expression system in
lung epithelial cells, p15Ink4b (a member of a different group of inhibitors that inhibits only cyclin D-dependent G1 kinases) binds to and inhibits the cyclin D-dependent kinases,
prevents p27 binding to these cdk complexes, and promotes p27 binding and inhibition
of cyclin-cdk2. In vitro, however, p15 prevents p27 binding only if it first has access to
cyclin D-cdk4. The different subcellular location of p15
and p27 ensures the prior access of p15 to cdk4. In the cell, p15 is localized mostly in
the cytoplasm, whereas p27 is nuclear. p15 prevails over p27 or a p27 construct
consisting of the cdk inhibitory domain tagged with a nuclear localization signal.
However, when p15 and p27 are forced to reside in the same subcellular location (either the cytoplasm or the nucleus), p15 no longer prevents p27 from binding to cdk4.
These properties allow p15 and p27 to coordinately inhibit cdk4 and cdk2 (Reynisdóttir, 1997).
The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively
inefficient. This has led to the suggestion that assembly might be a regulated step. The CDK inhibitors p21CIP, p27KIP, and p57KIP2 all promote the association of cdk4
with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or
cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex
increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a
simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic
studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase association constant, respectively, mostly
because of a decrease in disassociation constant. At low concentrations, p21 promotes the assembly of active kinase
complexes, whereas at higher concentrations, it inhibits activity. Immunodepletion
experiments demonstrate that most of the active cdk4-associated kinase activity also associates with
p21. To confirm these results in a natural setting, the assembly of endogenous complexes was examined
in mammary epithelial cells after release from a G0 arrest. Cyclin D1
and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This
complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity.
Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase
activity is also p21 associated. All three CIP/KIP inhibitors are found to target cdk4 and cyclin
D1 to the nucleus. It has been suggested that in addition to their roles as inhibitors, the p21 family of proteins,
originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program
kinase complexes for specific functions. Although all three of the inhibitors behave similarly with respect to promoting complex assembly, only p21 promotes the assembly of complexes with active RB kinase activity. Under the same conditions as those used for p21, both p27 and p57 cause only a gradual loss of cdk4-associated RB kinase activity. Possibly p27 and p57 may act only as inhibitors, revealing some fundamental difference between the way they interact with cdk/cyclin D complexes and the way p21 interacts (LaBaer, 1997).
Although p27(Kip1) has been considered a general inhibitor of G1 and S phase cyclin-dependent
kinases, the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4,
is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27
level completely inhibits Cdk2 and cell cycle progression. In contrast, the same or a larger increase in
p27 levels does not inhibit Cdk4 or its homolog Cdk6, despite extensive binding to these kinases. A
p27-cyclin A-Cdk2 complex formed in vitro is essentially inactive, whereas a p27-cyclin D2-Cdk4
complex is active as a retinoblastoma kinase and serves as a substrate for the Cdk-activating kinase
Cak. High concentrations of p27 inhibit cyclin D2-Cdk4, apparently through the conversion of active
complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential
interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 are similarly inhibited by bound p21(Cip1/Waf1).
The role of cyclin A-Cdk2 as a p27 target, and the role of cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes (Blain, 1997).
Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established
biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro; this inhibition is
believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells.
However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant
negative interference, is unable to arrest the cell cycle of the same cells. Functional differences have now been determined between p16 and Cdk4(N158).
p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16
dissociates cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) forms complexes with
cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 forms complexes with cyclin
E-Cdk2, and Cdk2-associated kinase activities are correspondingly inhibited. Cells engineered to express moderately
elevated levels of cyclin E become resistant to p16-mediated growth suppression. These results demonstrate that inhibition of
cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of
cyclin E-dependent kinases is required in p16-mediated growth suppression (Jiang, 1998).
It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells are observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlates with the presence of functional pRb. G2 arrest is more prominent in pRb-negative cells. The arrest distribution does not correlate with the p53 status, and the proliferating-cell nuclear antigen (PCNA) binding activity of p21 does not appear to be involved, since p27, which lacks a PCNA binding domain, produces a similar arrest profile. In addition, DNA endoreduplication occurs in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in cells arrested in G2 by p21. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. The tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway (Niculescu, 1998).
The predominant G2 arrest observed in pRb-negative cells and limited G2 arrest observed in pRb-positive cells is most likely due to p21-mediated inhibition of cyclin A-Cdk2. The dynamics of arrest in pRb-negative cells probably reflects the inefficiency of inhibition of cyclin E-Cdk2. It is unlikely that inhibition of cyclin B1-associated kinase by p21 contributes to G2 arrest, since there is no evidence for significant amounts of cyclin B1 associated with p21. Nevertheless, it has been observed that in p21-induced G2-arrested cells, cyclin B1-Cdk2 is inhibited by phosphorylation of Cdc2. Although the mechanism for this indirect inhibition by p21 is not yet known, a similar phenomenon has been observed in Xenopus egg extracts, where inhibition of Cdk2 leads to inhibitory phosphorylation of Cdc2 and concomitant G2 arrest. pRb is necessary to block endoreduplication. After arrest in G2, a significant subpopulation of pRb-negative cells responding to p21 or p27 expression undergo cycles of endoreduplicative DNA replication. Although a significant fraction of pRb-positive cells arrests in G2, endoreduplication is never observed. Two conclusions can be drawn from these observations: (1) p21 can arrest cells in G2 in a physiological environment that is permissive for entering the S phase without an intervening mitosis. This would seem to be a violation of the normal safeguards that prevent cell cycle events from occurring out of order. (2) Initiation of the S phase, however, whether from G1 or G2, requires neutralization of the inhibitory functions of pRb. This cannot happen in the presence of both pRb and p21. The incomplete inhibition of cyclin E- and/or cyclin A-associated kinase activities may allow sufficient activity for initiation of replication but not sufficient to proceed to mitosis. The appropriate balance may not be met in every cell, since many apparently do not undergo endoreduplication. Licensing of origins may not be efficient, since endoreduplicative replication appears to proceed slowly. But the fact that pRb appears to be capable of blocking endoreduplication in G2-arrested cells suggests a role in enforcing the order of cell cycle events. Whatever critical functions downstream of pRb are required for replication after passage through G1 appear to be required again if replication is to occur from G2. The regulation of pRb phosphorylation, in fact, may be an important G2 function of p21 in response to DNA damage (Niculescu, 1998).
A variant form of p27 was unexpectedly detected in a synchronized culture of NIH3T3 cells treated with serum. The expression levels of this form of p27, which lacks its amino (NH2)-terminal region, reach their maximum during G2/M phase.
Since the appearance of the NH2-terminal truncated form of p27 coincides with increased expression of Cdc2, it was
hypothesized that p27 may play a role in regulating Cdc2 catalytic activity. To test this hypothesis, wild type p27, as well as the
amino-terminal (Np27) and carboxyl-terminal (Cp27), were individually expressed, purified, and examined for their ability to
regulate CDC2 kinase activity in vitro. Both p27 and Np27 inhibit CDC2 kinase activity. In
marked contrast to this, Cp27 enhances the CDC2 kinase activity. Cp27 and p27 are
phosphorylated by CDC2, whereas Np27 is not. Deletion of the putative CDC2
phosphorylation site in the carboxyl-terminal domain of Cp27 diminishes activation of CDC2 kinase activity otherwise
stimulated by Cp27. A similar deletion does not have any effect on the inhibitory function of p27. Together these results suggest
that the carboxyl-terminal domain of p27 may activate CDC2 kinase activity in vivo during G2/M and that this effect may be
regulated by serine/threonine phosphorylation (Uren, 1997).
In human and murine cells, there are three known Cdc25 proteins: Cdc25A, Cdc25B, and Cdc25C (Drosophila homolog: String). The three phosphatases share approximately 40 to 50% homology. Cdc25C and Cdc25A function at G2-M and G1-S transitions during the human cell cycle, respectively. Cdc25A associates with,
dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27
are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals
(such as p53) and transforming growth factor beta (TGF-beta). A
cyclin binding motif has been identified near the N terminus of Cdc25A that is similar to the cyclin binding
Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the
catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin
E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the
dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21
competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the
dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the
dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene
up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from
inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts
from inhibition by p21. These results describe a mechanism by which the Myc- or
Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive
pathways counterbalance each other by competing for cyclin-cdks (Saha, 1997).
Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase
activities are Cdk inhibitors -- proteins that are induced in response to various antiproliferative signals but that can also
oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes
containing the prototype Cdk inhibitor p21 transit between active and inactive states: Cdk complexes associated
with one p21 molecule remain active until they associate with additional p21 molecules. However, using a number of
different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes, it has been demonstrated
unambiguously that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain
only one stably bound inhibitor molecule. Even phosphorylated forms of p21 remain efficient inhibitors of Cdk
activities. Therefore the level of Cdk inactivation by p21 is determined by the fraction of kinase complexed with the
inhibitor and not by the stoichiometry of inhibitor bound to the kinase or the phosphorylation state of the Cdk
inhibitor (Hengst, 1998).
During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent
activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. After treatment with microtubule inhibitors (MTIs), cells
deficient in the Cdk inhibitor p21(Waf1/Cip1) enter S phase with a 4N DNA content or greater, a process known as
endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced
endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems:
1). HCT116 p21(+/+) and p21(-/-) cells and 2). H1299 cells containing an inducible p21 expression vector (HIp21).
Both HCT116 p21(-/-) cells and noninduced HIp21 cells endoreduplicate after MTI treatment. Analysis of
G1-phase Cdk activities demonstrates that the induction of p21 inhibits endoreduplication through direct
cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to
proliferating-cell nuclear antigen in HCT116 p21(+/+) cells parallels the onset of endoreduplication in
HCT116 p21(-/-) cells. In contrast, loss of p21 does not lead to deregulated cyclin D1-dependent kinase
activities, nor does p21 directly regulate cyclin B1/Cdc2 activity. MTI-induced
endoreduplication in p53-deficient HIp21 cells is due to levels of p21 protein below a threshold required for
negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restores cyclin E/Cdk2 regulation and
prevents endoreduplication. Based on these findings, it is proposed that p21 plays an integral role in the
checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects
in microtubule dynamics (Stewart, 1999).
Progression through the cell cycle is regulated in part by the sequential activation and inactivation of
cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK
inhibitors (CKIs). p27(Kip1) was first identified as a CKI that binds and inhibits cyclin A/CDK2
and cyclin E/CDK2 complexes in G1. p27 has an additional property, the ability to
induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic
destruction box. Other CKIs [p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)] do not induce cleavage
of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The
C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce
cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with
cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the
continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage
site, and blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting
complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating
that the p27-induced cleavage of cyclin A is part of a separate pathway. This protease is referred to as Tsap
(pTwenty-seven-activated protease) (Bastians, 1998).
Subcellular location of CDK inhibitors p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA)
through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear
localization. A novel cytoplasmic localization of p21(Cip1/WAF1) is reported in peripheral blood monocytes (PBMs) and in U937 cells
undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and the
biological consequences of this cytoplasmic expression have been analyzed. U937 cells, which exhibit nuclear p21(Cip1/WAF1), demonstrate G1 cell-cycle arrest
and subsequently differentiated into monocytes. The latter event is associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1),
concomitant with a resistance to various apoptogenic stimuli. Cytoplasmic p21(Cip1/WAF1) forms a
complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion
mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) does not induce cell cycle arrest nor monocytic
differentiation, but leads to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus,
cytoplasmic p21(Cip1/WAF1) itself acts as an inhibitor of apoptosis. These findings highlight the different functional roles of
p21(Cip1/WAF1); such roles are determined by the intracellular distribution of p21(Cip1/WAF1) and are dependent on the stage of differentiation (Asada, 1999).
The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the
mechanisms that regulate its intracellular transport are poorly understood. p27 is phosphorylated on Ser10 in vivo, and mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is
trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and
phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic
localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes (Rodier, 2001).
The cyclin-dependent kinase inhibitor, p27Kip1, which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27Kip1 is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. The kinase responsible for S10 phosphorylation has been identified as human kinase interacting stathmin (hKIS); it is shown to regulate cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27Kip1 and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G0/G1, and expression of hKIS overcomes growth arrest induced by p27Kip1. Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27-/- cells treated with KIS siRNA grow and progress to S/G2 similar to control treated cells, implicating p27Kip1 as the critical target for KIS. Through phosphorylation of p27Kip1 on S10, hKIS regulates cell cycle progression in response to mitogens (Boehm, 2002).
The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Data is presented indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. These data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, it was found that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, it is reported that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy (Chu, 2007).
p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 310-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases (Grimmler, 2007).
p27Kip1 is an intrinsically unstructured protein (IUP) that controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Usually, binding of p27 inactivates the kinase; however, p27 was surprisingly also found to be associated with active cyclin D holoenzymes. p27 level is frequently controlled by regulated translation and proteolysis. The protein is abundant in quiescent (G0) cells and is relatively stable in G0 and early G1 phase. p27 becomes unstable as cells progress toward S phase. p27 degradation is initiated by different ubiquitin ligases. Among these, the KPC1 complex ubiquitinates free unphosphorylated p27, whereas Skp2-dependent E3 ligase complexes target p27 only after phosphorylation on threonine 187 (T187). Active cyclin E/Cdk2 can phosphorylate T187 of cyclin/Cdk-bound p27. While free and active cyclin/Cdk2 efficiently phosphorylates Cdk-bound p27, p27-bound Cdk2 is catalytically inactive due to p27-mediated remodeling of the catalytic cleft and displacement of ATP. This has suggested that degradation by the SCF-Skp2 pathway may require p27-free cyclin E/Cdk2 and has led to the puzzle of how p27 degradation can be initiated in G1 (Grimmler, 2007).
This study reports that p27 can be phosphorylated on a tyrosine residue at position 88 (Y88) within its Cdk-binding domain. This phosphorylation caused the inhibitory 310-helix of p27 to be ejected from the ATP-binding pocket of Cdk2. Y88-phosphorylated p27 still binds to cyclin/Cdk complexes, but the associated kinase retains significant catalytic activity. Furthermore, Y88-phosphorylated p27 becomes an efficient substrate for phosphorylation on T187 by Cdk2 within the trimeric complex. Thus, Y88 phosphorylation may trigger p27 ubiquitination in the absence of free cyclin/Cdk2 and may initiate SCF-Skp2-dependent p27 degradation at the G1/S transition (Grimmler, 2007).
Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. This study shows that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because (A) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; (B) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; (C) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; (D) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at the Thr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth and migration (Zheng, 2010).
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