Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits cdk2 kinase, causing G1 arrest. Here, we have investigated if a p53 fusion protein is a substrate for cdk2 kinase in vitro. Cdk2 kinase was immunoprecipitated from NIH3T3 cells and allowed to phosphorylate a human p53-GST (glutathione-s-transferase) fusion protein. Cdk2 and cyclin E-cdk2 efficiently phosphorylated both wild-type (wt) and mutant p53-GST. Cdk2 immunoprecipitated from cells in Go and early G1 exhibited minimal p53 kinase activity, whereas cells in S-phase displayed high levels of p53 kinase activity. If NIH3T3 cells were X-ray irradiated to induce DNA damage, cdk2 p53 kinase activity was rapidly inhibited within 1 h, but had recovered by 4 h post irradiation. Mutation of serine 315 of p53 to alanine (p53-S315A) abolished phosphorylation by cdk2 kinase. However, wtp53 and p53-S315A were equally effective at activating transcription when cotransfected with a p53 reporter construct. The results demonstrate that ser 315 of p53 is phosphorylated by cdk2 in vitro. However, ser 315 of wtp53 is not required for transcriptional activity in vivo, suggesting that cdk2 phosphorylation of p53 may be involved in regulating other cellular functions of wtp53.
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PMID:Cdk2 kinase phosphorylates serine 315 of human p53 in vitro. 762 34

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
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PMID:Phosphorylation of E2F-1 by cyclin A-cdk2. 783 23

The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.
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PMID:Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes. 824 49

The E6 and E7 proteins of the high-risk human papillomaviruses (HPVs) act coordinately to immortalize human keratinocytes. These viral oncoproteins function by binding and altering the activity of cellular proteins which regulate cell cycle progression. Among the proteins bound by E7 are the retinoblastoma protein, Rb, as well as the related p107 and p130 proteins. In addition, E7 binds cyclin A, which regulates transit through the S and G2/M phases of the cell cycle. In this study, we demonstrate that HPV 18 E7 also associates with cyclin E which controls the G1/S transition. E7/cyclin E complexes were immunoprecipitated from E7-expressing cells as well as from cell extracts using GST-E7 fusion proteins. E7 was found to complex with a single form of cyclin E, and the binding was mediated through p107. Both E7/cyclin E and E7/cyclin A complexes exhibit kinase activity through associated cdk2 proteins which can contribute to phosphorylation of p107. The association of E7 with proteins which regulate transit through the cell cycle may provide an additional mechanism by which infection with human papillomaviruses results in cellular hyperproliferation.
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PMID:Human papillomavirus E7 oncoproteins bind a single form of cyclin E in a complex with cdk2 and p107. 855 88

The gene mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently (Savitsky et al., 1995a) and the complete coding sequence of this gene, ATM, has been reported (Savitsky et al., 1995b). The derived amino acid sequence demonstrates significant homologies to several proteins containing a phosphatidylinositol 3-kinase (PI3-kinase) domain, including the yeast TOR proteins and the human protein FRAP. Since the TOR and FRAP proteins are targets for the immunosuppressive drug rapamycin, we have investigated the effects of this compound on A-T cells. We report here that 3 A-T cell lines are more resistant than control cells to rapamycin's growth inhibiting effects but were more sensitive to the PI3-kinase inhibitor wortmannin. As expected rapamycin (1 nM) inhibited the rate of exit of control cells from G1 phase but failed to perturb the progression of A-T cells. This difference in cell cycle progress after rapamycin treatment is reflected in ribosomal S6 protein kinase (p70S6k) by both a downward mobility shift on SDS-PAGE and inhibition of activity. Furthermore, the G1 phase cyclin-dependent kinase, cyclin E-cdk2, was rapidly inhibited in control cells post-treatment, whereas in A-T cells it took considerably longer to observe inhibition. There was no evidence that a GST-FKBP12 fusion protein specifically precipitated the ATM protein in the presence of rapamycin in either cell type. These results demonstrate that the ATM protein is not a direct target for rapamycin but its functional loss renders cells more resistant to this compound.
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PMID:Rapamycin resistance in ataxia-telangiectasia. 880 86

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.
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PMID:Cyclin E, a redundant cyclin in breast cancer. 898 90

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.
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PMID:Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity. 918 31

P130 shares structural and functional homology with pRb and p107. One property common to p107 and p130, but not to pRb, is the ability to stably interact with cyclin A/cdk2 and cyclin E/cdk2 complexes in vitro and in vivo. Using GST-p130 fusion proteins representing various regions of p130, baculovirus-produced cyclin A/cdk2 and cyclin E/cdk2 complexes were found to interact with residues within a part of p130 known as the spacer region. Cyclin E was able to bind the p130 spacer region in the presence or absence of cdk2 whereas cyclin A binding was dependent upon the presence of cdk2. The smallest p130 fusion protein sufficient to interact with cyclin A/cdk2 or cyclin E/cdk2 complexes contained p130 amino acids 652-698 and deletion of p130 amino acids 680-682 abolished binding to both of the cyclin/cdk2 complexes. When overexpressed in C33A cells, a p130 mutant containing a deletion of amino acids 620-697 was unable to form complexes with either cyclin A or cyclin E. This p130 mutant was at least as active as wild type p130 in suppressing the growth of G418 resistant colonies when overexpressed in C33A or SAOS-2 cells.
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PMID:Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes. 918 54

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.
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PMID:BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases. 924 50

The cell cycle events accompanying TGF-beta1-induced growth arrest of normal mouse resting B lymphocytes stimulated by LPS were investigated. We showed that TGF-beta1 prevents the retinoblastoma protein (pRb) phosphorylation and induces growth arrest in mid- to late G1. To explore the molecular basis of the effect of TGF-beta1, we analyzed the in vitro kinase activities of cyclin/cyclin-dependent kinase (cdk) complexes involved in the progression through G1 phase and in the G1/S transition, by using the glutathione S-transferase-pRb fusion protein as a substrate. Cdk2-associated kinase activity was strongly induced in mitogen-treated B cells. It was dramatically inhibited by TGF-beta1 as were the cyclin E- and cyclin A-dependent kinase activities. TGF-beta1 treatment had no significant effect on the expression of two G1/S phase proteins, cyclin E and cdk2. In contrast, the appearance of cyclin A, occuring in late G1 phase, was almost totally inhibited by TGF-beta1. We also showed that expression of the cdk inhibitor protein p27Kip1 decreased as cells progressed through the G1 phase. An accumulation of p27 was found in TGF-beta1-treated cells, showing that TGF-beta1 prevented LPS-induced decline of p27. Finally we found that the lack of kinase activity associated with cyclin E/cdk2 complexes was correlated with increased amounts of cdk2- and cyclin E-bound p27. Overall, these results suggest that both cyclin A and cdk2 may be active participants in the TGF-beta1-induced cell cycle arrest in normal mouse B cells and indicate the involvement of p27 in this mechanism.
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PMID:Effect of TGF-beta1 on cell cycle regulatory proteins in LPS-stimulated normal mouse B lymphocytes. 937 8


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