SEARCH · PubMed Health

Results for “Constitutive activation”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1997

Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active G(S)-coupled receptors.

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active G(S)-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

Adenylyl Cyclases↗

PubMed1997

The 96 kDa protein kinase activated by oncogenic Ras in Xenopus egg extracts is also activated by constitutively active Mek: activation requires serine/threonine phosphorylation.

In the Xenopus egg and oocyte system, oncogenic Ras protein can induce cell cycle arrest. The effect of oncogenic Ras on the cell cycle seems to be mediated by the Raf-Mek-Erk pathway of Ras signal transduction since constitutively active Raf, Mek, or Erk can mimic the effect of oncogenic Ras protein and since specific inhibition of these kinases can block the effect of oncogenic Ras. Using activated Xenopus egg extracts, we previously reported that the cell cycle arrest induced by oncogenic Ras correlates with the activation of a 96 kDa protein that phosphorylates histone h2b in vitro. This result raised the possibility that the 96 kDa kinase (designated as p96h2bk) is a potential target of the Raf-Mek-Erk pathway that links the pathway to the control of the cell cycle. We report here that constitutively active Mek1 could activate p96h2bk in the absence of oncogenic Ras. Moreover, inhibition of endogenous Mek by a specific inhibitor, PD 098059, suppressed the activation of p96h2bk by oncogenic Ras. These results are consistent with the concept that p96h2bk is a component or target of the Raf-Mek-Erk pathway. Furthermore, we have shown that activation of p96h2bk requires serine/threonine phosphorylation of p96h2bk.

Animals↗

PubMed2001

Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.

Animals↗

PubMed1994

Regulation of cAMP-mediated gene transcription by wild type and mutated G-protein alpha subunits. Inhibition of adenylyl cyclase activity by muscarinic receptor-activated and constitutively activated G(o) alpha.

We have used a luciferase reporter gene under the transcriptional control of a cAMP response element (CRE) to monitor the effects of G-protein alpha subunits on cAMP-regulated gene expression and to examine muscarinic acetylcholine receptor (mAChR) functional coupling to G-proteins. Expression in JEG-3 cells of a mutationally activated Gi alpha-2 in which glutamine 205 is replaced with leucine (Q205L) decreased forskolin-stimulated expression from the CRE-luciferase gene by up to 75%. Similarly, mutation of glycine 43 (corresponding to glycine 12 in p21ras) to valine decreased forskolin-stimulated expression from the CRE-luciferase gene by a maximum of 50%, indicating that this mutation activates the G-protein and is potentially oncogenic. Transfection of the activated Q205L G(o) alpha subunit decreased forskolin stimulation of CRE-luciferase expression. Transfected wild type G(o) alpha was also able to couple the m4 mAChR receptor to inhibition of AC. The amino-terminal myristoylation site was removed from wild type Gi alpha-2 and Q205L Gi alpha-2 by changing glycine 2 to alanine (G2A). Gi alpha-2 with the G2A and Q205L mutations was unable to decrease forskolin stimulation of CRE-mediated luciferase activity. Furthermore, G2A Gi alpha-2 was unable to couple the m4 mAChR to inhibition of AC. Thus, myristoylation is required both for the function of constitutively active Q205L Gi alpha-2 and for receptor-mediated activation of wild type Gi alpha-2.

Adenylyl Cyclase Inhibitors↗

PubMed1999

A strategy to make constitutively active MAP kinase by fusing with constitutively active MAP kinase kinase.

Classical mitogen-activated protein kinases (MAPKs) play a pivotal role in a variety of cellular signal transduction pathways. MAPKs are activated by phosphorylation at specific threonine and tyrosine residues catalyzed by upstream MAPK kinases (MAPKKs). Mutations of these two activation phosphorylation sites into acidic amino acids, however, do not convert MAPKs into constitutively active forms. Here, we report an approach to make a molecule with constitutive MAPK activity. The nuclear export signal-disrupted, constitutively active MAPKK was fused to the N-terminal end of wild-type MAPK. When the resulting fusion protein was expressed in Escherichia coli, the MAPK moiety became phosphorylated and the fusion protein was constitutively active as MAPK. Moreover, when expressed in mammalian cultured cells, the fusion protein was also activated as MAPK and was able to induce marked morphological changes in NIH-3T3 cells. These results suggest that the fusion protein can work as constitutively active MAPK and that this approach may be applicable to other members of the MAPK family to make constitutively active forms.

3T3 Cells↗

PubMed2006

3D modeling of the activated states of constitutively active mutants of rhodopsin.

The activated (R*) states in constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) are presumably characterized by lower energies than the resting (R) states. If specific configurations of TM helices differing by rotations along the long transmembrane axes possess energies lower than that in the R state for pronounced CAMs, but not for non-CAMs, these particular configurations of TM helices are candidate 3D models for the R* state. The hypothesis was studied in the case of rhodopsin, the only GPCR for which experimentally determined 3D models of the R and R* states are currently available. Indeed, relative energies of the R* state were significantly lower than that of the R state for the rhodopsin mutants G90D/M257Y and E113Q/M257Y (strong CAMs), but not for G90D, E113Q, and M257Y (not CAMs). Next, the developed build-up procedure successfully identified few similar configurations of the TM helical bundle of G90D/M257Y and E113Q/M257Y as possible candidates for the 3D model of the R* state of rhodopsin, all of them being in good agreement with the model suggested by experiment. Since constitutively active mutants are known for many of GPCRs belonging to the large rhodopsin-like family, this approach provides a way for predicting possible 3D structures corresponding to the activated states of the TM regions of many GPCRs for which CAMs have been identified.

Amino Acid Substitution↗

PubMedDate not supplied

Modulation of the constitutive activated STAT3 transcription factor in pancreatic cancer prevention: effects of indole-3-carbinol (I3C) and genistein.

BACKGROUND: The signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor required in proliferation and differentiation. STAT3 is activated constitutively in a number of cancers. MATERIALS AND METHODS: This study was conducted to assess the possible involvement of STAT3 activation in pancreatic cancer and the potential for this pathway as a target in chemopreventive strategy. RESULTS: STAT3 was shown for the first time to be constitutively activated in human pancreatic carcinoma specimens but not in normal pancreatic tissues. Constitutively activated STAT3 was also found in pancreatic tumor cell lines (Panc-1 and MIA PaCa-2) which could be modulated by indole-3-carbinol (13C) and genistein. At concentrations higher than 10 microM, STAT3 constitutive activation is inhibited by both agents. Induction of apoptosis by 13C was also demonstrated. CONCLUSION: Given its critical role in tumorigenesis, our results suggest that STAT3 activation provides an important and appropriate target for chemoprevention in pancreatic cancer treatment.

Adenocarcinoma↗

PubMed2005

Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel activity through activation of PKC. This study indicates that constitutive cation channel activity in ear artery myocytes is mediated by DAG which is generated by PC-PLD via phosphatidic acid which represents a novel activation pathway of cation channels in vascular myocytes.

Animals↗

PubMed1997

Mechanistic aspects of estrogen receptor activation probed with constitutively active estrogen receptors: correlations with DNA and coregulator interactions and receptor conformational changes.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Role of upstream activation sequences and integration host factor in transcriptional activation by the constitutively active prokaryotic enhancer-binding protein PspF.

PspF, the transcriptional activator of the pspA operon of Escherichia coli, which belongs to the enhancer binding protein (EBP) family of sigma54 activator proteins, is constitutively active in an in vitro transcription assay. PspF protein, together with RNA polymerase holoenzyme containing sigma54, is required for in vitro transcription from the pspA promoter. EBP proteins are typically subject to regulation either by post-translational modification or interaction of a specific ligand with an N-terminal regulatory domain. However, unlike other members of the EBP family, PspF lacks this domain. pspA is positively regulated by IHF in vitro, and this regulation is dependent on the topology of the DNA; a linear template is much more dependent on IHF than a supercoiled template. EBP binding to upstream activating sequences (UAS) in their target promoters is mediated by the C-terminal domain which contains a helix-turn-helix DNA-binding motif. A mutant PspF protein lacking the C-terminal DNA-binding domain is active in vitro, although at much higher concentrations than the wild-type protein. In vitro transcription from pspA templates missing one or both of the UAS sites is reduced relative to wild-type templates, but is still appreciable; however, IHF acts as a negative regulator of pspA transcription on these mutant templates. Thus, PspF bound to non-specific sequences upstream of the pspA promoter can activate pspA transcription, but this activation is inhibited by IHF. These data, taken together, support the model that a precise promoter geometry is necessary for IHF to positively regulate transcription and that IHF may act to prevent activation from inappropriately spaced upstream sites.

Bacterial Proteins↗

PubMed2003

Generation and characterization of highly constitutive active histamine H3 receptors.

Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wild-type (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and Ki values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased Ki values against the MT6 receptors (2.5- to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor.

Animals↗

PubMed2006

Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants.

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) are associated with clinical responsiveness to gefitinib in patients with non-small-cell lung cancers (NSCLC). Recently, we have identified many novel EGFR mutations in NSCLC tissues. In this study, we found that gefitinib could suppress the tyrosine phosphorylation of most EGFR mutants better than the wild-type receptor. However, gefitinib had quite variable growth-suppressive effects on different EGFR mutant-expressing cells. All tested EGFR mutants have high basal phosphorylation at multiple tyrosine residues. Upon EGF stimulation, the mutated EGFRs did not have apparently stronger phosphorylation at tyrosines 845, 992, 1,068, and 1,173 than the wild-type receptor. However, stronger phosphorylation at tyrosine 1,045 was observed in the S768I, L861Q, E709G, and G719S mutants. The E746-A750 deletion mutant was less responsive to EGF than the wild-type and other mutant receptors. The S768I, L861Q, E709G, and G719S mutants were refractory to EGF-induced ubiquitination and had more sustained tyrosine phosphorylation. E709G and G719S also lacked EGF-induced receptor downregulation. Our results indicate that, in addition to sensitivity to gefitinib, EGFR mutations also caused various changes in EGFR's regulatory mechanisms, which may contribute to the constitutive activation of EGFR mutants and oncogenesis in NSCLC.

Animals↗

PubMed1999

The human vasoactive intestinal Peptide/Pituitary adenylate cyclase activating peptide receptor 1 (VPAC1): constitutive activation by mutations at threonine 343.

The human vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide receptor1 (VPAC1) belongs to the class II subfamily of G protein-coupled receptors. Specific changes by mutagenesis of a strictly conserved threonine (H) into lysine (K), proline (P) or alanine (A) at position 343 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells evoked a 3.5 fold-increase in the cAMP level as compared to cells transfected with the wild-type receptor. In contrast other mutants such as T343C, T343E or T343F were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the T343K mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants T343K-E36A and T343K-D68A were no longer constitutively activated. A control double mutant (T343K-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VPAC1 receptor can be evoked by specific mutations of T343 at the junction of the second intracellular loop and fourth transmembrane segment. This constitutive activation appears to require the functional integrity of the N-terminal extracellular VIP binding domain.

Animals↗

PubMed2003

Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function.

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure, but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.

Actins↗

PubMed2006

Comparison of homology models and X-ray structures of the nuclear receptor CAR: assessing the structural basis of constitutive activity.

The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity.

Amino Acid Sequence↗

PubMed1998

Creation of a constitutively activated state of the 5-HT2A receptor by site-directed mutagenesis: revelation of inverse agonist activity of antagonists.

Constitutively active GPCR have revealed novel properties of drugs that exhibit classical competitive antagonism at the native forms of GPCR. These drugs reverse basal levels of constitutive activity, indicating that they have inverse agonist activity. We were interested in determining if competitive antagonists of the native 5-HT2A receptor, in particular, antipsychotic drugs, exhibit inverse agonist activity at the constitutively active 5-HT2A receptor. All of the drugs tested reduced basal IP production of constitutively active 5-HT2A receptors, indicating that they all exhibited inverse agonist activity. Risperidone and ketanserin produced the greatest inhibition of basal IP production resulting in a reduction of basal activity in the C322K mutant receptor of 82% and 80%, respectively. Antipsychotic drugs display inverse agonist activity, indicating that stabilization of the inactive conformation of the 5-HT2A receptor may be a key component of their mechanism of action.

Amino Acid Substitution↗

PubMed2002

p53 regulates Stat3 phosphorylation and DNA binding activity in human prostate cancer cells expressing constitutively active Stat3.

Constitutive activation of the signal transducer and activator of transcription 3 (Stat3) and mutation of the p53 are both commonly detected in human prostate cancer cells. We sought to investigate whether there is functional regulation of Stat3 by wild-type (wt) p53. Our results demonstrate that expression of wt p53 but not mutant p53 significantly reduced tyrosine phosphorylation of Stat3 and inhibited Stat3 DNA binding activity in both DU145 and Tsu prostate cancer cell lines that express constitutively active Stat3. Expression of the p53 downstream target, p21(WAF-1), did not have any inhibitory effect on Stat3 phosphorylation. Wt p53 but not p21(WAF-1) induced dramatic apoptosis in these prostate cancer cells. Expression of wt p53 did not cause a reduction of phosphorylation-independent Stat3 protein and reduction of phosphorylation of three unrelated protein kinases, ERK1, ERK2 (ERK1/2), and AKT. Interestingly, p53-dependent apoptosis occurred in the presence of high levels of phosphorylated AKT and ERK1/2 in both DU145 and Tsu prostate cancer cells. Further, we evaluated a series of established human prostate, breast, and ovarian cancer cell lines and found that all cancer cell lines expressing constitutively active Stat3, only harbor mutated or deleted p53. One implication of these results is that the anti-proliferative activities of p53 may not be compatible with the constitutive Stat3 signal in cancer cells.

Adenocarcinoma↗

PubMed2004

Molecular dynamics simulations of the human CAR ligand-binding domain: deciphering the molecular basis for constitutive activity.

The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptors that function as ligand-activated transcription factors. CAR plays an essential role in the metabolism of xenobiotics and shows--in contrast to related receptors--constitutive activity. However, the molecular basis for the constitutive activity remains unclear. In the present study, homology models of the ligand binding domain (LBD) were generated based on the crystal structures of the related pregnane X (PXR) and the vitamin D receptor (VDR). The models were used to investigate the basal activity of CAR and the effect of coactivator binding. Molecular dynamics (MD) simulations of complexed and uncomplexed receptor revealed a hypothesis for the activation mechanism. The suggested mechanism is supported by experimental results from site-directed mutagenesis. The basal activity of CAR can be explained by specific van-der-Waals interactions between amino acids on the LBD and its C-terminal activation domain (AF-2). Docking studies with the GOLD program yielded the interaction modes of structurally diverse agonists, giving insight into mechanisms by which ligands enhance CAR activity.

Amino Acid Sequence↗