Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.     

A Casein kinase 1/Checkpoint kinase 1 pyrazolo-pyridine protein kinase inhibitor as novel activator of the p53 pathway

Reactivation of the wild-type p53 pathway is one key goal aimed at developing targeted therapeutics in the cancer research field. Although most p53 protein kinases form ‘p53-activating’ signals, there are few kinases whose action can contribute to the inhibition of p53, as Casein kinase 1 (CK1) and Checkpoint kinase 1 (CHK1). Here we report on a pyrazolo-pyridine analogue showing activity against both CK1 and CHK1 kinases that lead to p53 pathway stabilisation, thus having pharmacological similarities to the p53-activator Nutlin-3. These data demonstrate the emerging potential utility of multivalent kinase inhibitors.     

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6

The human papillomavirus (HPV) protein E6 can promote the ubiquitination of the p53 tumour suppressor in vitro, providing an explanation for the ability of E6 to induce p53 degradation in vivo and contribute to the potential tumorigenic effect of the virus. Instead, in non-infected cells, p53 levels are primarily destabilised by the ubiquitin E3 ligase activity of the Mdm2 protein. Here we have compared the effects of E6 and Mdm2 on p53 ubiquitination in vivo. We show that whereas in the presence of Mdm2 proteasome inhibitors induce the accumulation of ubiquitinated forms of p53, this does not occur in the presence of E6. Accordingly, we confirm that the effect of E6 and p53 is independent of the six C-terminal lysine residues in p53, which have previously been described to play an important role for effective ubiquitination and degradation of 53 mediated by Mdm2. We also show that other yet unidentified residues in p53 are also susceptible to ubiquitination. These results indicate that E6 does not induce ubiquitination of p53 in the same way as Mdm2 in order to promote its degradation, suggesting important differences between the Mdm2 and E6 effects on p53 degradation.     

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.     

Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1

Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.     

Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase

The serine/threonine kinase, Pim-1, appears to be involved in regulating proliferation, differentiation and cell survival of lymphoid and myeloid cells. In this study, we have found that amino acid residues 140-147 (RKRRQTSM) at the C-terminal end of p21(Cip1/WAF1), a cyclin-dependent kinase (CDK) inhibitor, constitute an ideal phosphorylation consensus sequence for Pim-1. We demonstrate that Pim-1 efficiently phosphorylates this peptide sequence as well as the p21 protein in vitro. We also demonstrate by pull-down assay and by immunoprecipitation that Pim-1 associates with p21. During phorbol ester-induced differentiation of U937 cells, both Pim-1 and p21 expression levels increase with Pim-1 levels increasing in both the nucleus and cytoplasm while p21 remains primarily cytoplasmic. Co-transfection of wild type p21 with wild type Pim-1 results in cytoplasmic localization of p21 while co-transfection of wild type p21 with kinase dead Pim-1 results in nuclear localization of p21. Consistent with the results from the phosphoamino acid assay, Pim-1 phosphorylates transfected p21 only on Thr(145) in p21-deficient human fibroblasts and this phosphorylation event results in the cytoplasmic localization of p21. These findings demonstrate that Pim-1 associates with and phosphorylates p21 in vivo, which influences the subcellular localization of p21.     

p27kip1 protein levels reflect a nexus of oncogenic signaling during cell transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.     

HCV核心蛋白、p53、Mdm2及Bcl-2在HCC中的表达意义

目的:探讨HCC组织中的核心蛋白、突变p53、Mdm2、Bcl-2的表达以及其相关性,探讨HCV核心蛋白是否有可能促进p53突变、Mdm2和Bel-2的表达.方法:收集手术切除HCC组织42例,采用免疫组织化学EnVision法检测HCC组织核心蛋白、突变p53、Mdm2、Bel-2的表达,用统计学方法及临床联系分析它们之间的关系.结果:C蛋白、p53、Mdm2和Bcl-2在HCC癌组织中的表达率分别为40.5%、47.6%、75.6%、83.3%;4组强度等级资料Kruskal.Wallis秩和检验H=19.01,差异性显著(p=0.000),Mann-Whimey U test:C蛋白和p53、Mdm2、Bcl-2蛋白问的P值分别0.43、0.009、0.00;C蛋白与突变p53、Bcl-2阳性强度两者间相关性检验P值分别为0.000、0.914,相关系数rs分别为0.67、0.08;突变p53与Mdm2、Bcl-2两者相关性检验P值为0.000、0.27,相关系数rp为0.72、0.32.结论:在HCV核心蛋白表达的HCC中核心蛋白可能促进野生型p53突变和表达;突变p53很可能促进Mdm2的表达;核心蛋白和突变p53都有可能促进Bcl-2蛋白的表达;HCV相关的HCC中这些蛋白的变化很可能促进肝细胞增殖或恶性生长.     

Steady States and Oscillations in the p53/Mdm2 Network

p53 is activated in response to events compromising the genetic integrity of a cell. Recent data show that p53 activity does not increase steadily with genetic damage but rather fluctuates in an oscillatory fashion (Lahav et al., Nature Genetics, 36, 147-150, 2004). Theoretical studies suggest that oscillations can arise from a combination of positive and negative feedbacks or from a long negative feedback loop alone. Both negative and positive feedbacks are present in the p53/Mdm2 network, but it is not known what roles they play in the oscillatory response to DNA damage. We developed a mathematical model of p53 oscillations based on positive and negative feedbacks in the p53/Mdm2 network. According to the model, the system reacts to DNA damage by moving from a stable steady state into a region of stable limit cycles. Oscillations in the model are born with large amplitude, which guarantees an all-or-none response to damage. As p53 oscillates, damage is repaired and the system moves back to a stable steady state with low p53 activity. The model reproduces experimental data in quantitative detail. We suggest new experiments for dissecting the contributions of negative and positive feedbacks to the generation of oscillations.     

The Mdm2 and p53 genes are conserved in the Arachnids

The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the Placazoan to man. In common with D.melanogaster and C.elegans, there is a single copy of the p53 gene in T.adhaerens, while in the vertebrates three p53-like genes can be found: p53 , p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C.elegans and D.melanogaster. However, it is present in the Placazoan and the presence of multiple distinct p53 genes in the Sea anemone N.vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53 like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53.     

Conservation of all three p53 family members and Mdm2 and Mdm4 in the cartilaginous fish

Analysis of the genome of the elephant shark (Callorhinchus milii), a member of the cartilaginous fishes (Class Chondrichthyes), reveals that it encodes all three members of the p53 gene family, p53, p63 and p73, each with clear homology to the equivalent gene in bony vertebrates (Class Osteichthyes). Thus, the gene duplication events that lead to the presence of three family members in the vertebrates dates to before the Silurian era. It also encodes Mdm2 and Mdm4 genes but does not encode the p19^Arf gene. Detailed comparison of the amino acid sequences of these proteins in the vertebrates reveals that they are evolving at highly distinctive rates, and this variation occurs not only between the three family members but extends to distinct domains in each protein.     

The oncogenic serine/threonine kinase Pim-1 phosphorylates and inhibits the activity of Cdc25C-associated kinase 1 (C-TAK1): a novel role for Pim-1 at the G2/M cell cycle checkpoint

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.