Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.
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PMID:Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase. 1273 Jan 99

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.
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PMID:UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. 1367 83

The CDK inhibitor p27 plays a pivotal role in controlling cell proliferation during development, and has been implicated in tumorigenesis. Previous studies have demonstrated changes in p27 protein expression, but not in mRNA levels, in human pituitary tumors. It seems probable that the fall in p27 is due to increased degradation through the ubiquitin-proteasome pathway. Skp2 (S-phase kinase-interacting protein) is a specific F-box protein that allows the recognition and binding of phosphorylated p27 to the ubiquitin complex. The aim of our study was thus to investigate the possible role of Skp2 in pituitary tumorigenesis. A total of 59 human pituitary samples, 7 normal and 52 adenomas, were assessed for transcriptional expression of Skp2; 51 pituitary samples were assessed for protein expression. Real-time RT-PCR was performed on cDNA of reverse-transcribed mRNA for relative quantification of the Skp2 transcript. Immunostaining was performed using mouse monoclonal anti-Skp2 antibody. Skp2 mRNA and protein was detectable in every sample studied. Our results showed no significant difference between the pituitary tumors and normal pituitary tissue in Skp2 mRNA or nuclear protein expression. Individual tumor types had similar mRNA expression and variable protein expression. However, samples with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 staining. Our data suggest that increased p27 degradation through the ubiquitin-proteasome pathway could be regulated in pituitary tumors by changes in Skp2 expression, although other factors probably also play a role.
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PMID:The expression of the F-box protein Skp2 is negatively associated with p27 expression in human pituitary tumors. 1455 71

The clinical usefulness of trastuzumab (Herceptin; Genentech, San Francisco, CA) in breast cancer treatment is limited by the rapid development of resistance. We previously reported that IGF-I signaling confers resistance to the growth-inhibitory actions of trastuzumab in a model system, but the underlying molecular mechanism remains unknown. We used SKBR3/neo cells (expressing few IGF-I receptors) and SKBR3/IGF-IR cells (overexpressing IGF-I receptor) as our experimental model. IGF-I antagonized the trastuzumab-induced increase in the level of the Cdk inhibitor p27(Kip1). This resulted in decreased association of p27(Kip1) with Cdk2, restoration of Cdk2 activity and attenuation of cell-cycle arrest in G(1) phase, all of which had been induced by trastuzumab treatment in SKBR3/IGF-IR cells. We also found that the decrease in p27(Kip1) induced by IGF-I was accompanied by an increase in expression of Skp2, which is a ubiquitin ligase for p27(Kip1), and by increased Skp2 association with p27(Kip1). A specific proteasome inhibitor (LLnL) completely blocked the ability of IGF-I to reduce the p27(Kip1) protein level, while IGF-I increased p27(Kip1) ubiquitination. This suggests that the action of IGF-I in conferring resistance to trastuzumab involves targeting of p27(Kip1) to the ubiquitin/proteasome degradation machinery. Finally, specific inhibitors of MAPK and PI3K suggest that the IGF-I-mediated reduction in p27(Kip1) protein level by increased degradation predominantly involves the PI3K pathway. Our results provide an example of resistance to an antineoplastic therapy that targets one tyrosine kinase receptor by increased signal transduction through an alternative pathway in a complex regulatory network.
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PMID:Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells. 1464 98

TGF-beta is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-beta causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-beta inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-beta treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-beta negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-beta. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-beta-treated cultured cells. In addition, TGF-beta treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF-beta-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-beta occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.
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PMID:Negative regulation of SCFSkp2 ubiquitin ligase by TGF-beta signaling. 1467 46

Estrogen receptor (ER) beta counteracts the activity of ERalpha in many systems. In agreement with this, we show in this study that induced expression of ERbeta in the breast cancer cell line T47D reduces 17beta-estradiol-stimulated proliferation when expression of ERbeta mRNA equals that of ERalpha. Induction of ERbeta reduces growth of exponentially proliferating cells with a concomitant decrease in components of the cell cycle associated with proliferation, namely cyclin E, Cdc25A (a key regulator of Cdk2), p45(Skp2) (a key regulator of p27(Kip1) proteolysis), and an increase in the Cdk inhibitor p27(Kip1). We also observed a reduced Cdk2 activity. These findings suggest a possible role for ERbeta in breast cancer and imply that ERbeta-specific ligands may reduce proliferation of ER-positive breast cancer cells through actions on the G(1) phase cell-cycle machinery.
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PMID:Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. 1474 18

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.
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PMID:Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation. 1500 27

Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
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PMID:Bioluminescent imaging of Cdk2 inhibition in vivo. 1512 51

Prostacyclin has many effects in the vasculature; one of the less well understood is the ability to block cell cycle progression through G(1) phase. We previously reported that the prostacyclin mimetic, cicaprost, selectively inhibits cyclin E-cyclin-dependent kinase-2 (Cdk2), and now we show that it acts by regulating the expression of Skp2, the F-box protein that targets p27(Kip1) for ubiquitin-mediated proteolysis. First, we show that cicaprost prevents the late G(1) phase down-regulation of p27(Kip1) and that the inhibitory effect of cicaprost on cyclin E-Cdk2 activity and S phase entry is eliminated by deleting p27(Kip1). Levels of the closely related Cdk2 inhibitor, p21(Cip1), are unaffected by cicaprost. Moreover, we show that cicaprost blocks the induction of Skp2 mRNA and that ectopic expression of a Skp2 cDNA overrides the effect of cicaprost on p27(Kip1) levels and S phase entry. Our data show that inhibition of F-box protein gene expression can underlie the effect of a potent antimitogen.
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PMID:Antimitogenesis linked to regulation of Skp2 gene expression. 1512 98

Although Skp2 has been thought to mediate the degradation of p27 at the G(1)-S transition, Skp2(-/-) cells exhibit accumulation of p27 in S-G(2) phase with overreplication. We demonstrate that Skp2(-/-)p27(-/-) mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2(-/-) mice entered the endoduplication cycle after mitogenic stimulation, whereas this phenotype was not apparent in Skp2(-/-)p27(-/-) mice. Cdc2-associated kinase activity was lower in Skp2(-/-) cells than in wild-type cells, and a reduction in Cdc2 activity was sufficient to induce overreplication. The lack of p27 degradation in G(2) phase in Skp2(-/-) cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G(2)-M progression by mediating the degradation of p27.
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PMID:Skp2-mediated degradation of p27 regulates progression into mitosis. 1513 Apr 91


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