Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Regulation of p53 by activated protein kinase C-delta during nitric oxide-induced dopaminergic cell death

Selective cell death of dopaminergic neurons in the substantia nigra is the major cause of Parkinson disease. Current evidence suggests that this cell death could be mediated by nitric oxide by-products such as nitrate and peroxynitrite. Because protein kinase C (PKC)-delta is implicated in apoptosis of various cell types, we studied its roles and activation mechanisms in nitric oxide (NO)-induced apoptosis of SN4741 dopaminergic cells. When cells were treated with sodium nitroprusside (SNP), a NO donor, endogenous PKC-delta was nitrated and activated. Immunoprecipitation revealed that p53 co-immunoprecipitated with PKC-delta and was phosphorylated at the 15th serine residue in SNP-treated cells. An in vitro kinase assay revealed that p53 was directly phosphorylated by SNP-activated PKC-delta. The p53 Ser-15 phosphorylation was suppressed in SNP-treated cells when the NO-mediated activation of PKC-delta was inhibited by rottlerin or (-)-epigallocatechin gallate. Within 3 h of p53 phosphorylation, its protein levels increased because of decreased ubiquitin-dependent proteosomal proteolysis, whereas the protein levels of MDM2, ubiquitin-protein isopeptide ligase, were down-regulated in a p53 phosphorylation-dependent fashion. Taken together, these results demonstrate that nitration-mediated activation of PKC-delta induces the phosphorylation of the Ser-15 residue in p53, which increases its protein stability, thereby contributing to the nitric oxide-mediated apoptosis-like cell death pathway. These findings may be expanded to provide new insight into the cellular mechanisms of Parkinson disease.     

Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.

We have identified the phosphorylation sites in monkey p53 as well as specific changes in the phosphorylation state of free and complexed forms of simian virus 40 (SV40) large T antigen (T) and monkey p53 isolate from SV40 lytically infected CV1 cells. Phosphopeptide analyses of free T and p53 (To and p53o) and complexed T and p53 (T+ and p53+) fractions indicated several quantitative increases in the specific phosphorylation of complexed forms of both proteins. The N terminus of monkey p53+ is phosphorylated at Ser-9, Ser-15, Ser-20, either Ser-33 or Ser-37, and at least one of Ser-90 to Ser-99. The C-terminal sites are Ser-315 and Ser-392. On comparing p53+ with p53o, we found that labeling of the two N-terminal phosphotryptic peptides encompassing residues 1 to 20 and 33 to 101 was increased fivefold and that Ser-315 was sevenfold more labeled than was Ser-392. When T+ was compared with To, the N-terminal peptide containing phosphorylation sites Ser-106 through Thr-124 was twofold more labeled, the peptide containing Ser-657 through Ser-679 was sixfold more labeled and contained up to four phosphorylated serine residues, and Ser-639 and Thr-701 appeared unchanged. Overall, T+ molecules appeared to contain 3.5 mol more of labeled phosphate than did To, with the N-terminal peptide appearing fully phosphorylated. The phosphopeptide patterns obtained for lytic T+ and To fractions were nearly identical to those found for wild-type SV40 T (stably complexed with mouse p53) and mutant 5080 T (defective for p53 binding) expressed in transformed C3H10T1/2 cells (L. Tack, C. Cartwright, J. Wright, A. Srinivasan, W. Eckhart, K. Peden, and J. Pipas, J. Virol. 63:3362-3367, 1989). These results indicate that increases in specific phosphorylation sites in both T+ and p53+ correlate with the association of T with p53. The enhanced phosphorylation state may be a consequence of complex formation between T and p53 or reflect an increased affinity of p53 for highly phosphorylated forms of T.     

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.     

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.     

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein beta

Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPbeta is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells.     

ATM mediates phosphorylation at multiple p53 sites, including Ser(46), in response to ionizing radiation

The p53 tumor suppressor protein preserves genome integrity by regulating growth arrest and apoptosis in response to DNA damage. In response to ionizing radiation (IR), ATM, the gene product mutated in ataxia telangiectasia, stabilizes and activates p53 through phosphorylation of Ser(15) and (indirectly) Ser(20). Here we show that phosphorylation of p53 on Ser(46), a residue important for p53 apoptotic activity, as well as on Ser(9), in response to IR also is dependent on the ATM protein kinase. IR-induced phosphorylation at Ser(46) was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, but not PD169316, a p38 MAPK inhibitor. p53 C-terminal acetylation at Lys(320) and Lys(382), which may stabilize p53 and activate sequence-specific DNA binding, required Ser(15) phosphorylation by ATM and was enhanced by phosphorylation at nearby residues including Ser(6), Ser(9), and Thr(18). These observations, together with the proposed role of Ser(46) phosphorylation in mediating apoptosis, suggest that ATM is involved in the initiation of p53-dependent apoptosis after IR in human lymphoblastoid cells.     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.