The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.     

p27Kip1 Metabolism: A Fascinating Labyrinth

The progression through the phases of cell division cycle is regulated by different cyclins and cyclin-dependent kinases (CDKs) complexes. Due to their key function, the activity of cyclin/CDK complexes is controlled by several mechanisms, including the inhibition by a number of proteins collectively defined CDK inhibitors or CKIs. Among the CKIs, p27Kip1 represents a protein of central activity for the control of several phenotypes, including proliferation, differentiation and malignant transformation. p27Kip1 belongs to the growing family of “natively unfolded”, “in¬trinsically disordered” or “intrinsically unstructured” proteins. The disorder proteins present a very large number of possible conformations that, after the binding, converge to a well-defined structure with an extraordinary affinity for the target. As matter of fact, the absence of a preexisting folding strongly facilitates p27Kip1 interaction with a number of targets. Until recently, p27Kip1 has been solely viewed as a nuclear protein with the function of modulating cyclin–CDK activity and hence, cell-cycle progression. However, exhaustive studies have now demonstrated that the protein plays additional roles outside of the nucleus, including, particularly, the control of cell motility. Thus, the cellular localization is of fundamental importance in p27Kip1 function. Accordingly, at least two different mechanisms of degradation, occurring either in the nucleus or in the cytosol, have been observed. Convincing evidences have demonstrated that p27Kip1 is a phosphoprotein showing at least 6-8 phosphorylatable residues. However, the precise functional roles of the phosphorylations and the identification of the kinases responsible for the post-synthetic modifications are still debated. In this brief review, we will report the Literature data that connect the post-synthetic modifications of p27Kip1 with its function, localization and metabolism. The picture that emerges demonstrates that several of the pieces of the CKI metabolism are still nebulous.     

Switching cyclin D-Cdk4 kinase activity on and off

The cyclin-cdks are master regulators of cell proliferation. These serine/threonine kinases are the motors that both start and stop the cell cycle in response to proliferative or antiproliferative signals. They phosphorylate substrates required to trigger orderly cell cycle progression, and thus their activity is tightly regulated in order to prevent inappropriate activation. One of the main interfaces between the extraceullar environment and the cell cycle machinery is the interaction of the cyclin-cdks with two families of stoichiometric cyclin kinase inhibitors (CKIs), the Ink4s and the Cip/Kips. As their name suggests, the CKIs have historically been considered negative regulators of the cyclin-cdks, responsible for rapidly and effectively turning off cyclin-cdk activity. However, the interaction of cyclin D-cdk4 with the Cip/Kip family, and with p27Kip1 in particular, appeared complex. In addition to its ability to inhibit cyclin D-cdk4, p27 appeared to be a required assembly factor for the complex, binding in a non-inhibitory mode at least some of the time. Whether p27 was a cyclin D-cdk4/6 inhibitor or not was controversial, and how it might switch between these two modes was unknown. Arguing for a two state mechanism, we have recently shown that p27 can be both a cdk4 bound-inhibitor and a bound-non-inhibitor, depending on the growth state of the cell. This perspective highlights the significance of this finding in terms of normal cell cycle progression and tumor development.     

A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells

BACKGROUND: The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably normal when grown in vitro. One explanation may be the operation of compensatory pathways that control CDK activity and cell proliferation when normal pathways are inactivated. We have used mice lacking the CKIs p21(Cip1) and p27(Kip1) to investigate this issue, specifically with respect to CDK regulation by mitogens. RESULTS: We show that p27 is the major inhibitor of Cdk2 activity in mitogen-starved wild-type murine embryonic fibroblasts (MEFs). Nevertheless, inactivation of the cyclin E-Cdk2 complex in response to mitogen starvation occurs normally in MEFs that have a homozygous deletion of the p27 gene. Moreover, CDK regulation by mitogens is also not affected by the absence of both p27 and p21. A titratable Cdk2 inhibitor compensates for the absence of both CKIs, and we identify this inhibitor as p130, a protein related to the retinoblastoma gene product Rb. Thus, cyclin E-Cdk2 kinase activity cannot be inhibited by mitogen starvation of MEFs that lack both p27 and p130. In addition, cell types that naturally express low amounts of p130, such as T lymphocytes, are completely dependent on p27 for regulation of the cyclin E-Cdk2 complex by mitogens. CONCLUSIONS: Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.     

Cell-cycle inhibitors: three families united by a common cause

In the cellular program leading to DNA synthesis, signals that drive cells into S-phase converge at the level of CDK activity. The products of at least three different gene families, Ink4, Cip/Kip and the pRb pocket-protein family, suppress S-phase entry. Ink4 proteins act by antagonizing the formation and activation of cyclin D-CDK4 complexes, of which the ultimate downstream target as related to S-phase entry appears to be pRb. Cip/Kip inhibitors impinge upon that pathway by inhibiting CDK2 kinases that participate in the inactivation of pRb and, like cyclin E, may also have roles independent of pRb. How the activities of these three classes of proteins are coordinated remains obscure. In recent years, development of mouse models has accelerated the elucidation of this complex network, showing roles that are sometimes cooperative and sometimes overlapping. We will discuss the interrelationships between Cip/Kip inhibitors and the components of the pRb pathway, and how their activities ultimately regulate cell proliferation.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

Strategic cell-cycle regulatory features that provide mammalian cells with tunable G1 length and reversible G1 arrest

Transitions between consecutive phases of the eukaryotic cell cycle are driven by the catalytic activity of selected sets of cyclin-dependent kinases (Cdks). Yet, their occurrence and precise timing is tightly scheduled by a variety of means including Cdk association with inhibitory/adaptor proteins (CKIs). Here we focus on the regulation of G1-phase duration by the end of which cells of multicelled organisms must decide whether to enter S phase or halt, and eventually then, differentiate, senesce or die to obey the homeostatic rules of their host. In mammalian cells, entry in and progression through G1 phase involve sequential phosphorylation and inactivation of the retinoblastoma Rb proteins, first, by cyclin D-Cdk4,6 with the help of CKIs of the Cip/Kip family and, next, by the cyclin E-Cdk2 complexes that are negatively regulated by Cip/Kip proteins. Using a dynamical modeling approach, we show that the very way how the Rb and Cip/Kip regulatory modules interact differentially with cyclin D-Cdk4,6 and cyclin E-Cdk2 provides to mammalian cells a powerful means to achieve an exquisitely-sensitive control of G1-phase duration and fully reversible G1 arrests. Consistently, corruption of either one of these two modules precludes G1 phase elongation and is able to convert G1 arrests from reversible to irreversible. This study unveils fundamental design principles of mammalian G1-phase regulation that are likely to confer to mammalian cells the ability to faithfully control the occurrence and timing of their division process in various conditions.     

Cyclin Proteolysis and CDK Inhibitors: Two Redundant Pathways to Maintain Genome Stability in Mammalian Cells

Cyclin-dependent kinases (CDKs) are regulated by cyclin proteolysis and CDK inhibitors (CKIs) during mitotic exit and G1 phase in yeast and Drosophila, and disruption of both regulatory pathways leads to genomic instability. Our study using mouse cell lines that constitutively express a stabilized mutant of cyclin A revealed that three CKIs, p21, p27, and Rb-related p107, are responsible for cyclin proteolysis-independent inactivation of CDK during mitotic exit and G1. Enforced expression of cyclin A in the cells lacking all three CKIs induced rapid tetraploidization. Thus, the redundant pathways consisting of cyclin proteolysis and CKIs control CDK activity during mitotic exit and contribute to maintenance of genome stability in mammalian cells.     

Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis, and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs) p21^Cip1/Waf1, p27^Kip1, and p57^Kip2 bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signaling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular-dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.     

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.     

Glucocorticoids Induce G1 as Well as S-Phase Lengthening in Normal Human Stimulated Lymphocytes: Differential Effects on Cell Cycle Regulatory Proteins

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P< 0.005), a delayed increase in the duration of S-phase (P< 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P< 0.002). The expression of CKIs, mainly of p27^Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27^Kip1increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27^Kip1might amplify the functional effects of dexamethasone on kinasic complexes.     

CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells

The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has?a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.     

Multiple functions of p27(Kip1) and its alterations in tumor cells: a review

Cyclin-dependent kinases (CDKs), together with cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. p27(Kip1) is a member of a family of CDK inhibitors (CDIs) that bind to cyclin/CDK complexes and arrest cell division. There is considerable evidence that p27(Kip1) plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis. Moreover, p27(Kip1) is a putative tumor-suppressor gene that appears to play a critical role in the pathogenesis of several human malignancies and its reduced expression has been shown to correlate with poor prognosis in cancer patients. This study reviews current information on the functions of p27(Kip1), its abnormalities found in human tumors, and the possible clinical implications of these findings with respect to the management of cancer patients.     

P-TEFb- the final frontier

Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27^Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57^Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21^Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development.