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.

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.

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.

Phosphorylation of the p53 tumour-suppressor protein at three N-terminal sites by a novel casein kinase I-like enzyme.

Wild-type mouse p53, expressed in Escherichia coli, was phosphorylated by highly purified casein kinase I (CKI) from rabbit muscle. The major site of phosphorylation in the p53 was identified as serine 6, which is known to be phosphorylated in vivo. Serines 4 and 9 were also phosphorylated. To determine whether CKI is likely to be a physiological p53 kinase, SV3T3 cell lysates were fractionated on a Mono Q column and assayed for p53 kinase and casein kinase activities. Four p53 kinase activities were detected, one of which co-purified with CKI activity. This p53 kinase (designated PK270) further co-purified with CKI on sucrose gradients and had a native molecular weight, like CKI, in the range of 35,000-45,000. However, PK270 was separated from the bulk of CKI activity on a phosvitin-Sepharose affinity column, and was therefore likely to be a CKI-related kinase. In support of these conclusions, phosphorylation of p53, by both CKI and PK270, was inhibited by a peptide corresponding to a consensus CKI target sequence, but not by a non-specific peptide. Moreover, phosphopeptide analyses of p53 phosphorylated by CKI or by PK270 gave similar results, indicating that these two kinases phosphorylate the same sites in p53.

The conformational change of a murine temperature-sensitive p53 protein is independent of a change in phosphorylation status.

Wild-type and mutant p53 proteins exhibit opposing activities in respectively suppressing and promoting tumour development. In a rat embryo fibroblast cell line transformed with a murine temperature-sensitive p53 gene, p53 functions as a oncogene at 37 degrees C and as a tumour suppressor at 32 degrees C [Michalovitz, D., Halevy, O. & Oren, M. (1990). Cell, 62, 671-680]. We have used this cell line to investigate whether this temperature-dependent switching of function involves changes in the phosphorylation of p53 protein. Monoclonal antibodies PAb246 and PAb240 were used to immunoprecipitate metabolically 32P-labelled p53 protein in the 'wild-type' or mutant conformation from cells grown at 32 degrees C or 37 degrees C. Tryptic phosphopeptide maps were prepared from the isolated 'wild-type' and mutant p53 proteins. At 32 degrees C and 37 degrees C phosphopeptide maps of the 'wild-type' and mutant protein were identical. This demonstrates that the temperature-dependent conformation change, and associated functional change, in the p53 protein does not involve a change in the state of phosphorylation.

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.

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.

Murine p53 is phosphorylated within the PAb421 epitope by protein kinase C in vitro, but not in vivo, even after stimulation with the phorbol ester o-tetradecanoylphorbol 13-acetate.

The p53 tumour suppressor protein is thought to play a major role in the defence of the cell against agents which damage DNA. p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro. In this report, we have examined the phosphorylation of murine p53 by protein kinase C (PKC). Phosphopeptide mapping, phosphoamino acid analysis and radiosequence analysis of p53 phosphorylated by PKC in vitro indicated that serine 370 and threonine 377 were the major targets for phosphorylation and suggested that serine 372 and threonines 365 and 371 were minor phosphorylation sites. Site-directed mutagenesis confirmed that residues 370-372, all of which lie within the epitope for monoclonal antibody PAb421, were phosphorylated in vitro. The p53 from 32P-labelled SV3T3 cells showed a phosphopeptide pattern which includes peptides with mobilities similar to those arising from phosphorylation of residues 370-372 by PKC in vitro. Only two of these in vivo-labelled phosphopeptides co-migrated in two dimensions with peptides labelled in vitro within the PAb421 epitope and their phosphorylation was not stimulated by the addition of the PKC activator o-tetradecanoylphorbol 13-acetate (TPA) to the cells, even though this treatment led to a fourfold stimulation of p53 phosphorylation by MAP kinase. Moreover, when the p53 proteins containing mutations at residues 370-372 were expressed in COS cells, there was no loss of any of the in vivo phosphopeptides, indicating that phosphorylation within the PAb42I epitope was undetectable in the cell. These data suggest that p53 and PKC may not interact in vivo. The two-dimensional migration pattern of the novel group of peptides is consistent with phosphorylation of previously uncharacterised sites within the central DNA binding region of p53.

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs.

The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.

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.

Modifications of p53 protein and accumulation of p21 and gadd45 mRNA in TGF-beta 1 growth inhibited cells.

Transforming growth factory beta (TGF-beta) is a potent growth inhibitor of epithelial cells. One of the strategies used to elucidate the anti-proliferative mode of action of TGF-beta is to find out whether the receptor-generated signals interact with components of the basic machinery of the cell cycle. In this study we examined whether p53 and two other cycle inhibitory genes that can be transactivated by p53 are affected by TGF-beta 1 in epithelial cells. We show that TGF-beta 1 signalling controls the intracellular localization as well as the phosphorylation pattern and the stability of p53 protein. TGF-beta signalling also elevates the expression of p21/waf-1 and gadd45. The observed modifications in the protein suggest that p53 is involved in mediation of TGF-beta 1 growth inhibition. However, in TGF-beta 1 growth inhibited cells, wild type p53 is not required for the accumulation of the two p53 downstream targets p21/waf-1 and gadd45.

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.

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 interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase.

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.

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.

New developments in the multi-site phosphorylation and integration of stress signalling at p53.

PURPOSE: To summarize recent progress in the understanding of the role of multi-site phosphorylation in mediating the integration of stress signals at the p53 tumour suppressor protein. RESULTS: The p53 protein plays a key role in the response to a range of cellular stresses including agents that can damage DNA; consequently the involvement of p53 in sensing these effects is central to the prevention of tumour development. p53 is a potent but latent transcription factor that can be activated by a range of cellular stresses leading to the induction of cellular growth arrest or controlled cell removal through apoptosis. Accordingly, p53 is under tight control and is subject to several levels of regulation including multi-site phosphorylation. Recent evidence has implicated individual phosphorylation events in the activation of p53 by different types of stress (e.g. ionizing radiation, UV and mitotic spindle damage). CONCLUSIONS: A picture is now emerging of the p53 protein as an integration point for stress signals. Different signals impinge on different domains of the protein and may cooperate in modulating the type of p53 response, depending on the nature of the incoming signal.

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.

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.