Tyrosine phosphorylation of clathrin heavy chain under oxidative stress

In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.     

Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66(shc) are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66(shc) in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O(2) or 5% O(2) atmospheres until senescence was reached. Fibroblasts cultured under 20% O(2) tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O(2) tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O(2) at the same PD. p53 mRNA gradually decreased in 20% O(2) cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66(shc) mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O(2) atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66(shc) in senescence.     

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Oxidative stress induces H2AX phosphorylation in human spermatozoa

H2AX phosphorylation occurs following the induction of DNA double strand breaks (DSBs), thus collaborating with many other proteins to mediate important biological functions in somatic cells. In human spermatozoa, the present study showed that H(2)O(2) induced H2AX phosphorylation in a time- and dose-dependent manner. Moreover, such effect could be abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Meanwhile, the neutral comet assay also revealed DSBs production in correlation with H2AX phosphorylation assessed by flow cytometry. Besides H2AX phosphorylation, two other collaborating proteins, Rad50 and 53BP1, were also generated in spermatozoa after H(2)O(2) exposure. However, unlike in somatic FL cells, there were no distinctive focuses, but rather a whole nuclei staining pattern of these three proteins in spermatozoa. Additionally, gammaH2AX (the phosphorylated form of H2AX) staining in spermatozoa persisted despite the fact of a decrease in the number of gammaH2AX foci in FL cells after H(2)O(2) removal. Collectively, these results demonstrate that oxidative stress can induce H2AX phosphorylation in human spermatozoa through DSB induction, and that gammaH2AX may be used as a sensitive, novel marker for such DSBs. Moreover, the surveillance system involving gammaH2AX, Rad50, and 53BP1 in human spermatozoa cannot function effectively in DNA repair, but this system may possess other biological functions in response to DSBs.     

HDM2 negatively affects the Chk2-mediated phosphorylation of p53

By GST pull downs and co-immunoprecipitation analyses we found that recombinant Chk2 and HDM2 can form stable complexes in vitro. Chk2/HDM2 complexes were also detected in transfected Cos-1 cells over-expressing both proteins. Furthermore, we show that HDM2, as would be expected, severely affects the Chk2-catalyzed phosphorylation of p53. HDM2 itself is only slightly phosphorylated by Chk2. However, whereas HDM2 inhibits the Chk2-catalyzed p53 phosphorylation, HDM2 phosphorylation by Chk2 doubles in the presence of p53. The significance of the HDM2 phosphorylation is unknown, but it is possible that it might influence the stability of the HDM2/p53 complex.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma     

Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo

The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.     

Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress

Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H₂O₂) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H₂O₂-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H₂O₂ treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.     

Avian Reovirus activates a novel proapoptotic signal by linking Src to p53

We have previously shown that avian reovirus (ARV) S1133 and its structural protein σC cause apoptosis in cultured Vero cells through an unknown intracellular signaling pathway. This work investigates how ARV S1133 induces proapoptotic signals. Upon ARV S1133 infection and subsequent apoptosis, levels of p53 mRNA and protein, and p53 serine-46 and serine-392 phosphorylation increased. In addition, p53-driven reporter activity and levels of the p53-induced apoptotic protein bax were increased, and Src tyrosine-418 phosphorylation was elevated. UV-inactivated virus failed to activate Src, p53 or induce apoptosis. Over-expression of dominant negative p53, or treatment with tyrosine kinase inhibitor genistein protected cells from ARV S1133-induced apoptosis. Inhibition of Src by over-expression of C-terminal Src kinase (Csk) or treatment with Src family tyrosine kinase inhibitor SU-6656 diminished the ARV S1133-induced p53 expression, activation, and apoptosis. Over-expression of σC resulted in the upregulation of p53, p53 serine-46 phosphorylation, p53-driven reporter activity and accumulation of bax. σC expression during ARV S1133 infection was concomitant with the onset of apoptosis. These studies provide strong evidence that the viral gene expression is required for ARV S1133 to initiate a proapoptotic signal via Src to p53. In addition, σC was able to utilize a p53-dependent pathway to elicit apoptosis.     

Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser³⁹²-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser³⁹²-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser³⁹²-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser³⁹² presents an alternative for HCC chemotherapy. [BMB Reports 2014; 47(4): 221-226]     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.