Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27^Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27^Xic1 is expressed in the developing kidney in the nephrostomal regions. Using overexpression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27^Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27^Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27^Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27^Xic1, and reveal its differentiation function is not universally utilised in all developing tissues.     

Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.     

The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus

We have investigated the role of the cyclin-dependent kinase inhibitor, p27(Xic1), in the coordination of cell cycle exit and differentiation during early neurogenesis. We demonstrate that p27(Xic1) is highly expressed in cells destined to become primary neurones and is essential for an early stage of neurogenesis. Ablation of p27(Xic1) protein prevents differentiation of primary neurones, while overexpressing p27(Xic1) promotes their formation. p27(Xic1) may enhance neurogenesis by stabilising the bHLH protein, neurogenin. Moreover, the ability of p27(Xic1) to stabilise neurogenin and enhance neurogenesis localises to an N-terminal domain of the molecule and is separable from its ability to inhibit the cell cycle.     

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27^Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27^Xic1 is expressed in the developing kidney in the nephrostomal regions. Using over-expression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27^Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27^Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27^Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27^Xic1, and reveal its differentiation function is not universally utilised in all developing tissues.     

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.     

Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.     

Coupling Cell Cycle Exit,Neuronal Differentiation and Migration in Cortical Neurogenesis

The generation of new neurons in the cerebral cortex requires that progenitor cells leave the cell cycle and activate specific programs of differentiation and migration. Genetic studies have identified some of the molecules controlling these cellular events, but how the different aspects of neurogenesis are integrated into a coherent developmental program remains unclear. One possible mechanism implicates multifunctional proteins that regulate, both cell cycle exit and cell differentiation 1. A prime example is the cyclin-dependent kinase inhibitor p27Kip1, which has recently been shown to function beyond cell cycle regulation and promote both neuronal differentiation and migration of newborn cortical neurons, through distinct and separable mechanisms. p27Kip1 is therefore part of a machinery that couples the multiple events of neurogenesis in the cerebral cortex.     

N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts

N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin-dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin-mediated signals involved in myogenesis, we investigated whether N-cadherin-dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin-dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin-mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for beta-catenin accumulation at cell-cell contact sites. We propose that cell-cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.     

Inhibition of myogenesis by depletion of the glycogen-associated regulatory subunit of protein phosphatase-1 in rat skeletal muscle cells

In this study, we examined the role of the glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1(G)) in L6 rat skeletal muscle cell myogenesis. The level of PP-1(G) was depleted by transfection with an inducible antisense-oriented PP-1(G) gene. Western blot analysis of the PP-1(G)-depleted cell line revealed a >90% depletion of PP-1(G) protein and a 45% reduction in cellular PP-1 activity and abolished the ability of L6 myoblasts to differentiate into multinucleated myotubes. PP-1(G)-depleted cells also exhibited a marked reduction in the expression of the differentiation marker myogenin as well as creatine kinase. After 7 days in culture, PP-1(G)-depleted cells sustained myoblast levels of inhibitor of differentiation-2, whereas control L6 cells had a severely lower inhibitor of differentiation-2 level and progressed into myotubes. Myoblasts were unable to exit the cell cycle, as measured by the impaired induction of p27 cyclin-dependent kinase inhibitor, a >2-fold increase in DNA synthesis, and elevated levels of phosphorylated retinoblastoma protein (pRb). Replacement of the PP-1(G) gene restored PP-1(G) protein expression, PP-1 enzymatic activity, and the ability to differentiate into myotubes. We conclude that PP-1(G) plays a definite role in L6 myogenesis via its regulation of PP-1 catalytic activity.     

MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.     

The cyclin-dependent kinase inhibitors p15INK4B and p21CIP1 are critical regulators of fibrillar collagen-induced tumor cell cycle arrest

The extracellular matrix is a crucial component in determining cell fate. Fibrillar collagen in its native form inhibits cell proliferation, whereas in its monomeric form it stimulates proliferation. The observation of elevated levels of p27(KIP1) in cells plated in the presence of fibrillar collagen has led to the assumption that this kinase inhibitor was responsible for cell cycle arrest on fibrillar collagen. Here we provide evidence that p15(INK4b), rather than p27(KIP1), is the cyclin-dependent kinase inhibitor responsible for G0/G1 arrest of human melanoma cells grown on fibrillar collagen. Additionally, we demonstrate that fibrillar collagen can also arrest cells at the G2 phase, which is mediated in part by p21(CIP1). Our data, in addition to identifying cyclin-dependent kinase inhibitors important in cell cycle arrest mediated by fibrillar collagen, demonstrate the complexity of cell cycle regulation and indicate that modulating a single cyclin-dependent kinase inhibitor does not disrupt cell proliferation in the presence of fibrillar collagen.     

Myostatin regulates cell survival during C2C12 myogenesis

During the myogenic process in vitro, proliferating myoblasts withdraw irreversible from the cell cycle, acquire an apoptosis-resistant phenotype, and fuse into mature myotubes. The key factor regulating both myocyte cell cycle exit and viability during this transition is the the cyclin-dependent kinase inhibitor p21(cip1). Here we show that the expression of myostatin, a TGF-beta superfamily member known to act as a negative regulator of muscle growth, is upregulated in the course of C2C12 cells myogenesis. We also show that transient transfection of C2C12 myobasts with an expression vector encoding mouse myostatin cDNA efficiently inhibits cell proliferation. Paradoxically, myostatin cDNA overexpression also enhances the survival of differentiating C2C12 myocytes, probably by a mechanism involving, at least in part, upregulation of p21(cip1) mRNA. Our results suggest that myostatin role in myogenesis is more complex than initially suggested and involves another level of regulation apart from inhibition of myoblast proliferation.     

P27 knockout mice: reduced myostatin in muscle and altered adipogenesis

Knockout of the P27(kip) gene, which encodes a cyclin-dependent kinase inhibitor involved in cell proliferation regulation, results in growth enhancement in mice. To investigate how p27 deficiency affected adipogenesis and myogenesis, levels of PPARgamma, C/EBPalpha, and the myogenesis inhibitor, myostatin, were measured in p27(-/-) (n=14), p27(+/-) (n=18), and p27(+/+) mice (n=11). Body weight and gastrocnemius muscle (GC) mass were increased in p27(-/-) mice (P<0.05), but there were no differences in fat depot weights, percent body fat or serum leptin concentrations among genotypes. PPARgamma, but not C/EBPalpha, was markedly increased in p27(-/-) mice (P<0.05). There was also a higher incidence of inguinal fat apoptosis (P<0.01) in p27(-/-) mice. Myostatin levels were reduced in GC muscle of p27(-/-) mice (P<0.05). These findings suggest that in p27 deficient mice, increased skeletal muscle mass is mediated in part through decreased myostatin. Although total adiposity was not changed, increased PPARgamma levels suggest an alteration in adipogenesis.     

Proteolysis of <Emphasis Type="Italic">Xenopus</Emphasis> Cip-type CDK inhibitor,p16<Superscript>Xic2</Superscript>, is regulated by PCNA binding and CDK2 phosphorylation

Background

Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27^Xic1 (Xic1) which shares sequence homology with both p21^Cip1 and p27^Kip1 from mammals, p16^Xic2 (Xic2) which shares sequence homology with p21^Cip1, and p17^Xic3 (Xic3) which shares sequence homology with p27^Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4^Cdt2, little is known about the processes that regulate Xic2 or Xic3.

Methods

We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.

Results

Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.

Conclusions

During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.

     

Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4. Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation

Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.