Mutations of phosphorylation sites Ser10 and Thr187 of p27Kip1 abolish cytoplasmic redistribution but do not abrogate G0/1 phase arrest in the HepG2 cell line

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is an important regulator of cell cycle progression as it negatively regulates G(0/1) progression and plays a major role in controlling the cell cycle. The screening of the p27(Kip1) sequence identified many potential phosphorylation sites. Although Ser(10) and Thr(187) were shown to be important for p27(Kip1) function, the effects of a combined deletion of both sites on p27(Kip1) function are still unknown. To investigate the effects of the overexpression of exogenous p27(Kip1) protein lacking both the Ser(10) and Thr(187) sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27(Kip1) at Ser(10) and Thr(187) (S10A/T187A p27), and transfected into the HepG(2) cell line with Lipofectamine. Wild-type and mutant p27 plasmids S10A and T187A were transfected separately as control groups. As a result, the proliferation of HepG(2) cells was greatly inhibited and cell cycle was arrested in G(0/1) phase after exogenous p27(Kip1) double-mutant expression. All recombinant p27(Kip1) constructs were distributed in the nucleus after synchronization in G(0) phase by treatment with leptomycin B. The expressed wild-type and T187A p27(Kip1) proteins were translocated from the nucleus into cytoplasm when cells were exposed to 20% serum for 8 h, whereas the S10A p27(Kip1) and S10A/T187A p27(Kip1) proteins remained in the nucleus. FACS profiles and cell growth curves indicated that the Ser(10) and Thr(187) double mutant has no significant effect on the biological activities of cell cycle control and growth inhibition. Our results suggest that expression of the p27(Kip1) double-mutant abolishes its cytoplasmic redistribution but does not abrogate G(0/1) phase arrest in the HepG(2) cell line.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27Kip1

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We previously demonstrated that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in primary MKs in a PI3K dependent fashion. UT-7/TPO, a megakaryocytic cell line, stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to reduce p27Kip1 expression after TPO stimulation, and fail to induce p27Kip1 expression following TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In addition, the number of MKs is significantly increased in bone marrow from Foxo3a-deficient mice. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings indicate that the PI3K/Akt/FOXO3a/p27Kip1 pathway contributes to normal TPO-induced MK proliferation.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry

Cilia at the node generate a leftward fluid flow that breaks left-right symmetry. However, the molecular mechanisms that regulate ciliogenesis at the node are largely unknown. Here, we show that the epiblast-specific deletion of the gene encoding the BMP type 1 receptor (Acvr1) compromised development of nodal cilia, which results in defects in leftward fluid flow and, thus, abnormalities in left-right patterning. Acvr1 deficiency in mouse embryonic fibroblasts (MEFs) resulted in severe defects in their quiescence-induced primary cilia. Although the induction of quiescence in wild-type MEFs leads to an increase in the level of the cyclin-dependent kinase inhibitor p27(Kip1) and to rapid p27(Kip1) phosphorylation on Ser(10), MEFs deficient in Acvr1 show a reduction in both p27(Kip1) protein levels and in p27(Kip1) Ser(10) phosphorylation. The observed defects in cilium development were rescued by the introduction of p27(Kip1) into Acvr1-deficient MEFs, implying that BMP signaling positively controls p27(Kip1) stability in the G0 phase via p27(Kip1) Ser(10) phosphorylation, which is a prerequisite for induction of primary cilia. Importantly, in control embryos, p27(Kip1) protein is clearly present and strongly phosphorylated on Ser(10) in cells on the quiescent ventral surface of the node. By contrast, the corresponding cells in the node of Acvr1 mutant embryos were proliferative and showed a dramatic attenuation in both p27(Kip1) protein levels and phosphorylation on Ser(10). Our data suggest that cell quiescence controlled by BMP signaling via ACVR1 is required for transient formation of nodal cilia, and provide insight into the fundamental question of how the node represents the mechanistic `node' that regulates the development of left-right symmetry in vertebrates.     

Arginine vasopressin controls p27 protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27^Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27^Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27^Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27^Kip1 protein as well as phosphorylation of the p27^Kip1 protein at both Ser10 and Thr187.     

Phosphorylation of Skp2 regulated by CDK2 and Cdc14B protects it from degradation by APC(Cdh1) in G1 phase

The p27(Kip1) ubiquitin ligase receptor Skp2 is often overexpressed in human tumours and displays oncogenic properties. The activity of SCF(Skp2) is regulated by the APC(Cdh1), which targets Skp2 for degradation. Here we show that Skp2 phosphorylation on Ser64/Ser72 positively regulates its function in vivo. Phosphorylation of Ser64, and to a lesser extent Ser72, stabilizes Skp2 by interfering with its association with Cdh1, without affecting intrinsic ligase activity. Cyclin-dependent kinase (CDK)2-mediated phosphorylation of Skp2 on Ser64 allows its expression in mid-G1 phase, even in the presence of active APC(Cdh1). Reciprocally, dephosphorylation of Skp2 by the mitotic phosphatase Cdc14B at the M --> G1 transition promotes its degradation by APC(Cdh1). Importantly, lowering the levels of Cdc14B accelerates cell cycle progression from mitosis to S phase in an Skp2-dependent manner, demonstrating epistatic relationship of Cdc14B and Skp2 in the regulation of G1 length. Thus, our results reveal that reversible phosphorylation plays a key role in the timing of Skp2 expression in the cell cycle.     

Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization

In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.