The RNA helicase p68 (DDX5) is selectively required for the induction of p53-dependent p21 expression and cell-cycle arrest after DNA damage.

The RNA helicase p68 (DDX5) is an established co-activator of the p53 tumour suppressor that itself has a pivotal role in orchestrating the cellular response to DNA damage. Although several factors influence the biological outcome of p53 activation, the mechanisms governing the choice between cell-cycle arrest and apoptosis remain to be elucidated. In the present study, we show that, while p68 is critical for p53-mediated transactivation of the cell-cycle arrest gene p21(WAF1/CIP1), it is dispensable for induction of several pro-apoptotic genes in response to DNA damage. Moreover, p68 depletion results in a striking inhibition of recruitment of p53 and RNA Pol II to the p21 promoter but not to the Bax or PUMA promoters, providing an explanation for the selective effect on p21 induction. Importantly, these findings are mirrored in a novel inducible p68 knockout mouse model in which p68 depletion results in a selective inhibition of p21 induction in several tissues. Moreover, in the bone marrow, p68 depletion results in an increased sensitivity to γ-irradiation, consistent with an increased level of apoptosis. These data highlight a novel function of p68 as a modulator of the decision between p53-mediated growth arrest and apoptosis in vitro and in vivo.

Soil-test N recommendations augmented with PEST-optimized RZWQM simulations.

Improved understanding of year-to-year late-spring soil nitrate test (LSNT) variability could help make it more attractive to producers. We test the ability of the Root Zone Water Quality Model (RZWQM) to simulate watershed-scale variability due to the LSNT, and we use the optimized model to simulate long-term field N dynamics under related conditions. Autoregressive techniques and the automatic parameter calibration program PEST were used to show that RZWQM simulates significantly lower nitrate concentration in discharge from LSNT treatments compared with areas receiving fall N fertilizer applications within the tile-drained Walnut Creek, Iowa, watershed (>5 mg NL(-1) difference for the third year of the treatment, 1999). This result is similar to field-measured data from a paired watershed experiment. A statistical model we developed using RZWQM simulations from 1970 to 2005 shows that early-season precipitation and early-season temperature account for 90% of the interannual variation in LSNT-based fertilizer N rates. Long-term simulations with similar average N application rates for corn (Zea mays L.) (151 kg N ha(-1)) show annual average N loss in tile flow of 20.4, 22.2, and 27.3 kg N ha(-1) for LSNT, single spring, and single fall N applications. These results suggest that (i) RZWQM is a promising tool to accurately estimate the water quality effects of LSNT; (ii) the majority of N loss difference between LSNT and fall applications is because more N remains in the root zone for crop uptake; and (iii) year-to-year LSNT-based N rate differences are mainly due to variation in early-season precipitation and temperature.

Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53.

The p53 tumour suppressor is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation and proteasome-mediated degradation of p53. Many agents that induce p53 are inhibitors of transcription, suggesting that the p53 pathway can detect a signal(s) arising from transcriptional malfunction. Mdm2 associates with TAFII250, a component of the general transcription factor TFIID. Inactivation of TAFII250 in ts13 cells, which express a temperature-sensitive mutant of TAFII250, leads to the induction of p53 and cell cycle arrest. In the present study, we show that TAFII250 stimulates the ubiquitylation and degradation of p53 in a manner that is dependent upon Mdm2 and requires its acidic domain. Mechanistically, TAFII250 downregulates Mdm2 auto-ubiquitylation, leading to Mdm2 stabilization, and promotes p53-Mdm2 association through a recently defined second binding site in the acidic domain of Mdm2. These data provide a novel route through which TAFII250 can directly influence p53 levels and are consistent with the idea that the maintenance of p53 turnover is coupled to the integrity of RNA polymerase II transcription.

c-Abl phosphorylates Hdm2 at tyrosine 276 in response to DNA damage and regulates interaction with ARF.

The p53 tumour-suppressor protein is tightly regulated through its association with the Hdm2 E3 ligase. Activation of p53 by DNA strand breaks is orchestrated by the ataxia-telangiectasia mutated (ATM) protein kinase and involves interruption of Hdm2-mediated p53 degradation. As part of this mechanism ATM itself, and the ATM-activated protein tyrosine kinase, c-Abl, inhibit Hdm2 function through phosphorylation of serine 395 and tyrosine 394 (Y394), respectively. In the present study, we have identified a novel target of c-Abl in the Hdm2 protein, tyrosine 276 (Y276). We show that c-Abl phosphorylates this residue in vitro and confirm that Y394 is a target of c-Abl. We also show that Y276 is phosphorylated in a c-Abl-dependent manner in cultured cells and provide evidence that Y276 is phosphorylated in response to DNA damage coincident with the activation of c-Abl. Finally, we show that Y276 phosphorylation stimulates interaction with ARF, leading to increased levels of nucleolar Hdm2 and decreased turnover of p53. These data establish Y276 as a physiological target of c-Abl that contributes functionally to the induction of p53.

Agricultural practices influence flow regimes of headwater streams in western Iowa.

Agricultural tillage influences runoff and infiltration, but consequent effects on watershed hydrology are poorly documented. This study evaluated 25 yr (1971-1995) hydrologic records from four first-order watersheds in Iowa's loess hills. Two watersheds were under conventional tillage and two were under conservation (ridge) tillage, one of which was terraced. All four watersheds grew corn (Zea mays L.) every year. Flow-frequency statistics and autoregressive modeling were used to determine how conservation treatments influenced stream hydrology. The autoregressive modeling characterized variations in discharge, baseflow, and runoff at multi-year, annual, and shorter time scales. The ridge-tilled watershed (nonterraced) had 47% less runoff and 36% more baseflow than the conventional watershed of similar landform and slope. Recovery of baseflow after drought was quicker in the conservation watersheds, as evidenced by 365-d moving average plots, and 67% greater baseflow during the driest 2 yr. The two conventional watersheds were similar, except the steeper watershed discharged more runoff and baseflow during short (<30 d), wet periods. Significant multi-year and annual cycles occurred in all variables. Under ridge-till, seasonal (annual-cycle) variations in baseflow had greater amplitude, showing the seasonality of subsurface contaminant movement could increase under conservation practices. However, deviations from the modeled cycles of baseflow were also more persistent under conservation practices, indicating baseflow was more stable. Indeed, flow-frequency curves showed wet-weather discharge decreased and dry-weather discharge increased under conservation practices. Although mean discharge increased in the conservation watersheds, variance and skewness of daily values were smaller. Ridge tillage with or without terraces increased stream discharge but reduced its variability.

Nitrate leaching to subsurface drains as affected by drain spacing and changes in crop production system.

Subsurface drainage is a beneficial water management practice in poorly drained soils but may also contribute substantial nitrate N loads to surface waters. This paper summarizes results from a 15-yr drainage study in Indiana that includes three drain spacings (5, 10, and 20 m) managed for 10 yr with chisel tillage in monoculture corn (Zea mays L.) and currently managed under a no-till corn-soybean [Glycine max (L.) Merr.] rotation. In general, drainflow and nitrate N losses per unit area were greater for narrower drain spacings. Drainflow removed between 8 and 26% of annual rainfall, depending on year and drain spacing. Nitrate N concentrations in drainflow did not vary with spacing, but concentrations have significantly decreased from the beginning to the end of the experiment. Flow-weighted mean concentrations decreased from 28 mg L(-1) in the 1986-1988 period to 8 mg L(-1) in the 1997-1999 period. The reduction in concentration was due to both a reduction in fertilizer N rates over the study period and to the addition of a winter cover crop as a "trap crop" after corn in the corn-soybean rotation. Annual nitrate N loads decreased from 38 kg ha(-1) in the 1986-1988 period to 15 kg ha(-1) in the 1997-1999 period. Most of the nitrate N losses occurred during the fallow season, when most of the drainage occurred. Results of this study underscore the necessity of long-term research on different soil types and in different climatic zones, to develop appropriate management strategies for both economic crop production and protection of environmental quality.

Using the late spring nitrate test to reduce nitrate loss within a watershed.

Excessive nitrate leaching from the U.S. Corn Belt has created serious water quality problems and contributed to the expansion of the hypoxic zone in the Gulf of Mexico. We evaluated the effect of implementing the late spring nitrate test (LSNT) for corn (Zea mays L.) grown within a 400-ha, tile-drained subbasin in central Iowa. Surface water discharge and NO3 concentrations from the treated subbasin and two adjacent subbasins receiving primarily fall-applied, anhydrous ammonia were compared. In two of four years, the LSNT method significantly reduced N fertilizer applications compared with the farmers' standard practices. Average corn yield from LSNT fields and nonlimiting N fertilizer check strips was not significantly different. Autoregressive (AR) models using weekly time series in surface water NO3 concentration differences between the LSNT and control subbasins indicated no consistent significant differences during the pre-LSNT (1992-1996) period. However, by the second year (1998) of the treatment period (1997-2000), NO3 concentrations in surface water from the treated subbasin were significantly lower than the concentrations coming from both control basins. Annual average flow-weighted NO3 concentrations for the last two years (1999-2000) were 11.3 mg N L(-1) for the LSNT and subbasin and 16.0 mg N L(-1) for the control subbasins. Based on these values and the AR models, widespread adoption of the LSNT program for managing N fertilizer where fall N application is typically practiced could result in a > or = 30% decrease for NO3 concentrations in surface water.

Evaluation of nitrate nitrogen fluxes from a tile-drained watershed in central Iowa.

Nitrate N fluxes from tile-drained watersheds have been implicated in water quality studies of the Mississippi River basin, but actual NO3-N loads from small watersheds during long periods are poorly documented. We evaluated discharge and NO3-N fluxes passing the outlet of an Iowa watershed (5134 ha) and two of its tile-drained subbasins (493 and 863 ha) from mid-1992 through 2000. The cumulative NO3-N load from the catchment was 168 kg ha(-1), and 176 and 229 kg ha(-1) from the subbasins. The outlet had greater total discharge (1831 mm) and smaller flow-weighted mean NO3-N concentration (9.2 mg L(-1)) than the subbasins, while the larger subbasin had greater discharge (1712 vs. 1559 mm) and mean NO3-N concentration (13.4 vs. 11.3 mg L(-1)) than the smaller subbasin. Concentrations exceeding 10 mg L(-1) were common, but least frequent at the outlet. Nitrate N was generally not diluted by large flows, except during 1993 flooding. The outlet showed smaller NO3-N concentrations at low flows. Relationships between discharge and NO3-N flux showed log-log slopes near 1.0 for the subbasins, and 1.2 for the outlet, considering autocorrelation and measurement-error effects. We estimated denitrification of subbasin NO3-N fluxes in a hypothetical wetland using published data. Assuming that temperature and NO3-N supply could limit denitrification, then about 20% of the NO3-N would have been denitrified by a wetland constructed to meet USDA-approved criteria. The low efficiency results from the seasonal timing and NO3-N content of large flows. Therefore, agricultural and wetland best management practices (BMPs) are needed to achieve water quality goals in tile-drained watersheds.

Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover.

The p53 tumour suppressor protein is a short-lived transcription factor that becomes stabilized in response to a wide range of cellular stresses. Ubiquitination and the targeting of p53 for degradation by the proteasome are mediated by Mdm2 (mouse double minute clone 2), a negative regulatory partner of p53. Previous studies have suggested that DNA-damage-induced phosphorylation of p53 at key N-terminal sites has a pivotal role in regulating the interaction with Mdm2 but the precise role of phosphorylation of serines 15 and 20 is still unclear. Here we show that replacement of serine 15 and a range of other key N-terminal phosphorylation sites with alanine, which cannot be phosphorylated, has little effect on the ubiquitination and degradation of full-length human p53. In contrast, replacement of serine 20 makes p53 highly sensitive to Mdm2-mediated turnover. These results define distinct roles for serines 15 and 20, two sites previously demonstrated to be dependent on phosphorylation through mechanisms mediated by DNA damage and ATM (ataxia telangiectasia mutated). We also show that the polyproline region of p53, a domain that has a key role in p53-induced apoptosis, exerts a critical influence over the Mdm2-mediated turnover of p53.

Nitrate loss in subsurface drainage as affected by nitrogen fertilizer rate.

The relationships between N fertilizer rate, yield, and NO3 leaching need to be quantified to develop soil and crop management practices that are economically and environmentally sustainable. From 1996 through 1999, we measured yield and NO3 loss from a subsurface drained field in central Iowa at three N fertilizer rates: a low (L) rate of 67 kg ha(-1) in 1996 and 57 kg ha(-1) in 1998, a medium (M) rate of 135 kg ha(-1) in 1996 and 114 kg ha(-1) in 1998, and a high (H) rate of 202 kg ha(-1) in 1996 and 172 kg ha(-1) in 1998. Corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] were grown in rotation with N fertilizer applied in the spring to corn only. For the L treatment, NO3 concentrations in the drainage water exceeded the 10 mg N L(-1) maximum contaminant level (MCL) established by the USEPA for drinking water only during the years that corn was grown. For the M and H treatments, NO3 concentrations exceeded the MCL in all years, regardless of crop grown. For all years, the NO3 mass loss in tile drainage water from the H treatment (48 kg N ha(-1)) was significantly greater than the mass losses from the M (35 kg N ha(-1)) and L (29 kg N ha(-1)) treatments, which were not significantly different. The economically optimum N fertilizer rate for corn was between 67 and 135 kg ha(-1) in 1996 and 114 and 172 kg ha(-1) in 1998, but the net N mass balance indicated that N was being mined from the soil at these N fertilizer levels and that the system would not be sustainable.

Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization.

Mammalian casein kinase 1delta (CK1delta) is a homologue of the S. cerevisiae Hrr25p protein kinase. Hrr25p is involved in regulating diverse events including vesicular trafficking, gene expression, DNA repair, and chromosome segregation. In contrast to Hrr25p, little is known about the function, regulation, or subcellular localization of CK1delta. In the present study, we show that CK1delta in mammalian cells is mainly cytoplasmic and enriched within the Golgi and/or ER-Golgi transport vesicles, consistent with a role in vesicular trafficking. Transient expression of green fluorescent protein (GFP)- or FLAG peptide-tagged CK1delta showed localization similar to that of the endogenous CK1delta. GFP-CK1delta was also enriched at the centrosomes in interphase cells. Strikingly, two inactive mutant CK1delta proteins (K38M and T176I) showed almost exclusive nuclear staining, suggesting that protein kinase activity is required for normal localization of CK1delta and prevention of nuclear accumulation. The nuclear export inhibitor leptomycin B promoted nuclear enrichment of CK1delta indicating that nuclear localization of CK1delta occurs physiologically. Both endogenous CK1delta and GFP-CK1delta are enriched on the spindle poles in mitotic cells, consistent with a role in regulating spindle formation. Localization is a property of the protein kinase domain and is independent of the C-terminal noncatalytic domain. These data are consistent with roles for CK1delta in mammalian cells analogous to those of its yeast counterparts.

IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects.

The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been proposed to be involved in regulating DNA repair and chromosomal segregation. Recently, we showed that CK1delta localizes to the spindle apparatus and the centrosomes in cells with mitotic failure caused by DNA-damage prior to mitotic entry. We provide here evidence that 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), a novel inhibitor of CK1delta and CK1epsilon, triggers the mitotic checkpoint control. At low micromolar concentrations IC261 inhibits cytokinesis causing a transient mitotic arrest. Cells containing active p53 arrest in the postmitotic G1 phase by blockage of entry into the S phase. Cells with non-functional p53 undergo postmitotic replication developing an 8N DNA content. The increase of DNA content is accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images show that at low concentrations IC261 leads to centrosome amplification causing multipolar mitosis. Our data are consistent with a role for CK1delta and CK1epsilon isoforms in regulating key aspects of cell division, possibly through the regulation of centrosome or spindle function during mitosis.

Multiple sites of in vivo phosphorylation in the MDM2 oncoprotein cluster within two important functional domains.

The MDM2 oncoprotein is a negative regulatory partner of the p53 tumour suppressor. MDM2 mediates ubiquitination of p53 and targets the protein to the cytoplasm for 26S proteosome-dependent degradation. In this paper, we show that MDM2 is modified in cultured cells by multisite phosphorylation. Deletion analysis of MDM2 indicated that the sites of modification fall into two clusters which map respectively within the N-terminal region encompassing the p53 binding domain and nuclear export sequence, and the central acidic domain that mediates p14(ARF) binding, p53 ubiquitination and cytoplasmic shuttling. The data are consistent with potential regulation of MDM2 function by multisite phosphorylation.

The role of p53 in the response to mitotic spindle damage.

The p53 tumour suppressor protein has defined roles in G1/S and G2/M cell cycle checkpoints in response to a range of cellular stresses including DNA damage, dominant oncogene expression, hypoxia, metabolic changes and viral infection. In addition to these responses, p53 can also be activated when damage occurs to the mitotic spindle. Initially, spindle damage activates a p53-independent checkpoint which functions at the metaphase-anaphase transition and prevents cells from progressing through mitosis until the completion of spindle formation. Cells eventually escape from this block (a process termed 'mitotic slippage'), and an aberrant mitosis ensues in which sister chromatids fail to segregate properly. After a delay period, p53 responds to this mitotic failure by instituting a G1-like growth arrest, with an intact nucleus containing 4N DNA, but without the cells undergoing division. Cells lacking wild-type p53 are still able to arrest transiently at mitosis, and also fail to undergo division, underscoring that the delay in mitosis is p53-independent. However, these cells are not prevented from re-entering the cell cycle and can reduplicate their DNA unchecked, leading to polyploidy. Additionally, p53-null cells which experience spindle failure often show the appearance of micronuclei arising from poorly segregated chromosomes which have decondensed and been enclosed in a nuclear envelope. The ability of p53 to prevent their formation suggests an additional G2 involvement which prevents nuclear breakdown prior to mitosis. The molecular mechanism by which p53 is able to sense mitotic failure is still unknown, but may be linked to the ability of p53 to regulate duplication of the centrosome, the organelle which nucleates spindle formation.

Phosphorylation of murine p53, but not human p53, by MAP kinase in vitro and in cultured cells highlights species-dependent variation in post-translational modification.

The p53 tumour suppressor protein is tightly regulated by protein-protein association, protein turnover and a variety of post-translational modifications. Multisite phosphorylation plays a major role in activating and in finely tuning p53 function. The proline rich domain of murine p53 is a substrate for phosphorylation, in vitro and in cultured cells, by the p42ERK2 and p44ERK1 mitogen-activated protein (MAP) kinases. However, to date there have been no reports of attempts to determine whether p53 from any other species is a substrate for MAP kinase. In this paper we confirm that murine p53 is targeted by recombinant MAP kinase and by MAP kinases in extracts of both murine and human cells. In contrast, human p53 is not a substrate for recombinant MAP kinase nor are there any detectable levels of protein kinase activity in stimulated human cell extracts which phosphorylate the proline rich domain of human p53 in vitro. Finally, although stimulation of murine fibroblasts with o-tetradecanolylphorbol 13-acetate (TPA), an indirect activator of the MAP kinase pathway, leads to site-specific phosphorylation of murine p53, similar treatment of human fibroblasts and epithelial cells showed no significant changes in the phosphorylation pattern. These data are consistent with accumulating evidence that significant species-dependent differences exist in the post-translational modification of p53.

Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15.

p53 is a potent transcription factor which is regulated by sequential multisite phosphorylation and acetylation. In this paper, we identify threonine 18 of p53, a key site in regulating the interaction between p53 and its regulatory partner MDM2, as a novel site phosphorylated in vitro by purified recombinant casein kinase 1 (CK1) delta. Strikingly, phosphorylation of threonine 18 is dependent upon prior phosphorylation of serine 15. These data highlight an additional and physiologically important target residue for CK1 in p53 and suggest a potential mechanism by which sequential modification of a pivotal N-terminal residue in p53 may occur following stress-activated modification of serine 15.

Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2.

The p53 tumour suppressor protein is a labile transcription factor that is activated and stabilized in response to a wide range of cellular stresses, through a mechanism involving disruption of its interaction with MDM2, a negative regulatory partner. Induction of p53 by DNA damage additionally involves a series of phosphorylation and acetylation modifications, some of which are thought to regulate MDM2 binding. Here we report the effects of introducing mutations at several known or putative N-terminal phosphorylation sites on the transactivation function of p53. These studies highlight phosphorylation of Ser15, a key phosphorylation target during the p53 activation process, as being critical for p53-dependent transactivation. Biochemical data indicate that the mechanism by which phosphorylation of Ser15 stimulates p53-dependent transactivation occurs through increased binding to the p300 coactivator protein. The data also indicate that Ser15-dependent regulation of transactivation is independent of any involvement in modulating MDM2 binding, and that Ser15 phosphorylation alone is not sufficient to block the p53-MDM2 interaction.

Mechanisms of switching on p53: a role for covalent modification?

The p53 protein plays a pivotal role in activating and integrating adaptive cellular responses to a wide range of environmental stresses. Activation of p53 can occur by different molecular routes, depending on the nature of the activating signal. Central to the activation process, by whichever route, is the destabilization of the p53-MDM2 interaction. The molecular mechanisms which activate p53 involve elements of post-translational modification, protein stabilization and protein-protein interaction. Two central themes are emerging from recent work in this area. The first is that there are common events in the p53 activation process among different activating pathways. The second is that activation involves not just a single molecular event such as disruption of the p53-MDM2 interaction, but a series of sequential events the nature of which is governed by the type of activating stimulus. This review summarizes our current knowledge of the p53 activation process in response to two stimuli, DNA damage and activated oncogenes, and considers the contribution made by multisite phosphorylation in determining the nature of the p53 response.

Multisite phosphorylation and the integration of stress signals at p53.

The p53 tumour suppressor protein is a potent transcription factor that plays a major role in the defence against tumour development. p53 exists in a latent form that can be activated by a range of stresses including DNA damage, hypoxia, cytokines, metabolic changes, viral infection, and activated oncogenes. Activation of p53 can lead to cellular growth arrest prior to entry into either S phase or mitosis or can trigger cell death through apoptosis. The modification of p53 by multisite phosphorylation provides a potential link between stress signalling and the regulation of p53 activity, and there is now striking evidence that agents that activate p53 can lead to selective changes in its phosphorylation status. Topologically, the phosphorylation sites in p53 fall into two discrete functional domains. Four phosphorylation events take place within the N-terminal 83 amino acids containing the transactivation domain and a region involved in transcription-independent growth suppression. At least three of these modifications occur in response to agents that cause cellular stress such as DNA damage. At the C-terminus, there are three phosphorylation events, each of which can independently regulate the specific DNA-binding function of p53, suggesting convergent control by different signalling pathways. The multiplicity of these covalent modifications and their responsiveness to a wide range of signals suggest that p53 activity is tightly and coordinately controlled in response to stresses and changes in the cellular environment.