Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a search for effectors and targets of UVB signaling in mammalian cells, we screened a keratinocyte cDNA library with differentially subtracted UVB-enriched cDNA probes. One of the UVB induced cDNA clones proved to be the rat p21Cip1/WAF1 homologue. UVB irradiation caused a rise in p53 protein levels, in association with induction of p21Cip1/WAF1 and cyclin G expression. The effects of UVB irradiation induced p21Cip1/WAF1 on the cell cycle were examined. In contrast to gamma irradiation, which caused G2 arrest, UVB treatment of asynchronous neonatal rat keratinocytes (NK) led to a marked inhibition of replicative DNA synthesis and prolonged G1 and S phase arrests, persisting to 18-24 h, with recovery of cycling by 36 h post-UVB. G1 arrest was accompanied by inhibition of cyclin D-, E- and A-associated kinases. Kinase inhibition was not due to reduction in cyclin or cdk proteins. While the association of cyclin E with Cdk2 was moderately reduced, cyclin D1/Cdk4 and cyclin A/Cdk2 complexes were not disrupted. The activating threonine 160 phosphorylation of Cdk2 in cyclin complexes was not inhibited. An incremental binding of p21 with Cdk4 paralleled the inhibition of cyclin D1/Cdk4 kinase and a similar rise in Cdk2 binding to p21 was associated with inhibition of cyclin E and cyclin A dependent kinases. Furthermore, a rise in measurable p21Cip1/WAF1-Cdk2 inhibitory activity paralleled the loss of G1 cyclin-dependent kinase activity, supporting a role for p21Cip1/WAF1 in the UVB-induced checkpoints.
 ...
PMID:UVB radiation induces p21Cip1/WAF1 and mediates G1 and S phase checkpoints. 862 54

The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.
 ...
PMID:Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction. 863 17

Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
 ...
PMID:Cyclin-dependent kinase-2 (Cdk2) forms an inactive complex with cyclin D1 since Cdk2 associated with cyclin D1 is not phosphorylated by Cdk7-cyclin-H. 864 86

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.
 ...
PMID:Analysis of wild-type and mutant p21WAF-1 gene activities. 865 54

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.
 ...
PMID:Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. 867 31

Mutations of the BRCA1 gone in humans are associated with predisposition to breast and ovarian cancers. We show here that Brca1+/- mice are normal and fertile and lack tumors by age eleven months. Homozygous Brca1(5-6) mutant mice die before day 7.5 of embryogenesis. Mutant embryos are poorly developed, with no evidence of mesoderm formation. The extraembryonic region is abnormal, but aggregation with wild-type tetraploid embryos does not rescue the lethality. In vivo, mutant embryos do not exhibit increased apoptosis but show reduced cell proliferation accompanied by decreased expression of cyclin E and mdm-2, a regulator of p53 activity. The expression of cyclin-dependent kinase inhibitor p21 is dramatically increased in the mutant embryos. Buttressing these in vivo observations is the fact that mutant blastocyst growth is grossly impaired in vitro. Thus, the death of Brca1(5-6) mutant embryos prior to gastrulation may be due to a failure of the proliferative burst required for the development of the different germ layers.
 ...
PMID:The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. 867 8

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.
 ...
PMID:Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases. 875 45

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.
 ...
PMID:Cyclin-binding motifs are essential for the function of p21CIP1. 875 24

Fluoropyrimidines radiosensitize human colon cancer cells that progress into S phase in the presence of drug (M.A. Davis, H-Y. Tang, J. Maybaum, and T.S. Lawrence. Int. J. Radiat. Biol. 67. 509-512, 1995). We hypothesized that progression occurs in cells that generate elevated levels of cyclin E-dependent kinase activity despite the presence of the fluoropyrimidine. To test this hypothesis, we treated HT29 and SW620 human colon cancer cells with fluorodeoxyuridine under conditions that produced nearly complete inhibition of thymidylate synthase but which sensitized only the HT29 cells. We found that, whereas HT29 cells progressed into S phase and demonstrated increased cyclin E-dependent kinase activity, SW620 cells arrested just past the G1-S boundary and showed no change in kinase activity. Because these cell lines have the same p53 mutation, these findings suggest that there is a p53-independent G1-S checkpoint that mediates radiosensitization produced by fluorodeoxyuridine.
 ...
PMID:Fluoropyrimidine-mediated radiosensitization depends on cyclin E-dependent kinase activation. 876 7

Tumor suppressor p53 is a nuclear protein that is induced by DNA damage and is involved in G1 and G2 phase control of the cell cycle. p21WAF1/CIP1/SDI1 (p21), a cyclin-dependent kinase inhibitor, is a downstream target and effector of p53 to induce G1 arrest. Mimosine is a potent reversible late G1 phase blocker of the cell cycle. In this study, we showed that mimosine can increase both p21 mRNA and protein levels, indirectly inhibit cyclin E-associated kinase activity without affecting the cyclin E protein level, block human breast cancer cells (21PT) in the late G1 phase of the cell cycle, and induce a p53-independent p21 pathway in these cells. These results support the possibility of restoring a G1 checkpoint by use of mimosine. They also suggest that the mechanism of the effect of mimosine is complex and may have more than one target in the cell.
 ...
PMID:p21WAF1/CIP1/SDI1 is elevated through a p53-independent pathway by mimosine. 880 7


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>