Phosphorylated Amino Acid Research Articles

Differential gene expression in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice

BackgroundMore than 46 species of mammals can be naturally infected with Schistosoma japonicum in the mainland of China. Mice are permissive and may act as the definitive host of the life cycle. In contrast, rats are less susceptible to S. japonicum infection, and are considered to provide an unsuitable micro-environment for parasite growth and development. Since little is known of what effects this micro-environment has on the parasite itself, we have in the present study utilised a S. japonicum oligonucleotide microarray to compare the gene expression differences of 10-day-old schistosomula maintained in Wistar rats with those maintained in BALB/c mice.ResultsIn total 3,468 schistosome genes were found to be differentially expressed, of which the majority (3,335) were down-regulated (≤ 2 fold) and 133 were up-regulated (≥ 2 fold) in schistosomula from Wistar rats compared with those from BALB/c mice. Gene ontology (GO) analysis revealed that of the differentially expressed genes with already established functions or close homology to well characterized genes in another organisms, many are related to important biological functions or molecular processes. Among the genes that were down-regulated in schistosomula from Wistar rats, some were associated with metabolism, signal transduction and development. Of these genes related to metabolic processes, areas including translation, protein and amino acid phosphorylation, proteolysis, oxidoreductase activities, catalytic activities and hydrolase activities, were represented. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differential expressed genes indicated that of the 328 genes that had a specific KEGG pathway annotation, 324 were down-regulated and were mainly associated with metabolism, growth, redox pathway, oxidative phosphorylation, the cell cycle, ubiquitin-mediated proteolysis, protein export and the MAPK (mitogen-activated protein kinases) signaling pathway.ConclusionsThis work presents the first large scale gene expression study identifying the differences between schistosomula maintained in mice and those maintained in rats, and specifically highlights differential expression that may impact on the survival and development of the parasite within the definitive host. The research presented here provides valuable information for the better understanding of schistosome development and host-parasite interactions.

Phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is essential for human BK virus propagation in tissue culture

BK virus (BKV) infection may cause polyomavirus-associated nephropathy in patients with renal transplantation. Recently, the phosphorylated amino acids on the structural proteins VP1, VP2 and VP3 of BKV have been identified by liquid chromatography-tandem mass spectrometry in our laboratory. In this study, we further analysed the biological effects of these phosphorylation events. Phosphorylation of the BKV structural proteins was demonstrated by [(32)P]orthophosphate labelling in vivo. Site-directed mutagenesis was performed to replace all of the phosphorylated amino acids. The mutated BKV genomes were transfected into Vero cells for propagation analysis. The results showed that expression of the early protein LT and of the late protein VP1 by the mutants VP1-S80A, VP1-S80-133A, VP1-S80-327A, VP1-S80-133-327A and VP2-S254A was abolished. However, propagation of other mutants was similar to that of wild-type BKV. The results suggest that phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is crucial for BKV propagation.

Identification and characterization of microRNAs from Phaeodactylum tricornutum by high-throughput sequencing and bioinformatics analysis

BackgroundDiatoms, which are important planktons widespread in various aquatic environments, are believed to play a vital role in primary production as well as silica cycling. The genomes of the pennate diatom Phaeodactylum tricornutum and the centric diatom Thalassiosira pseudonana have been sequenced, revealing some characteristics of the diatoms' mosaic genome as well as some features of their fatty acid metabolism and urea cycle, and indicating their unusual properties. To identify microRNAs (miRNAs) from P. tricornutum and to study their probable roles in nitrogen and silicon metabolism, we constructed and sequenced small RNA (sRNA) libraries from P. tricornutum under normal (PT1), nitrogen-limited (PT2) and silicon-limited (PT3) conditions.ResultsA total of 13 miRNAs were identified. They were probable P. tricornutum-specific novel miRNAs. These miRNAs were sequenced from P. tricornutum under normal, nitrogen-limited and/or silicon-limited conditions, and their potential targets were involved in various processes, such as signal transduction, protein amino acid phosphorylation, fatty acid biosynthetic process, regulation of transcription and so on.ConclusionsOur results indicated that P. tricornutum contained novel miRNAs that have no identifiable homologs in other organisms and that they might play important regulator roles in P. tricornutum metabolism.

Theoretical Study of Bone Sialoprotein in Bone Biomineralization

Bone sialoprotein (BSP) is an acidic, non-collagenous protein specific to bone proposed previously to promote hydroxyapatite (HAP) nucleation and modulate HAP nanocrystal growth. Specifically, two phosphorylated acidic amino acid sequences in BSP, highly conserved across several vertebrates, are the proposed active sites. We selected one of these sites, i.e. (Sp)₂E₈, where Sp represents a phosphoserine as a model peptide to study the role of BSP. We used molecular dynamics simulations to determine whether an α-helix or a random coil peptide conformation promotes templated HAP nucleation. A bioinformatics method helps infer preferential crystal growth directions by predicting the likely peptide conformations adsorbed on the (001), (100), and (110) crystal faces of HAP. Results suggest that, independent of conformation, no stable nucleating template is formed and, thus, the ion distributions in the vicinity of the peptide that eventually lead to a stable nucleus start out with disordered arrangements of ions. When adsorbed on all three faces, the Sp residues bind strongly regardless of the peptide conformation, and the Glu residues show different propensities to form helical conformations. The lack of geometrical templating between the peptide residues and all HAP surface sites indicates that adsorption and subsequent crystal growth modulation may be structurally nonspecific.

Intermolecular Phosphoryl Transfer of N‐Phosphoryl Amino Acids

AbstractN‐Phosphoryl amino acids (NPAAs) are a novel series of N‐terminal‐activated amino acids that act as the energy source and phosphoryl donor in intra‐ and intermolecular phosphoryl transfer to form “high‐energy” species, such as acetyl phosphate and aminoacyl phosphates, and in the self‐assembled synthesis of polypeptides under mild aqueous conditions. In this work, the chemical reactivity of N‐mono(methoxyphosphoryl)glycine as a representative was investigated in detail by using a combination of the stable‐isotope‐labeling (15N) technique, 31P NMR, ESI‐MS/MS and LC‐MS. The phosphoryl group of NPAAs can be transferred intermolecularly to the carboxy group of another molecule through intramolecular cyclic pentacoordinate phosphoric–amino acid anhydride intermediates. In addition to C‐terminal activation by phosphate anhydride, amino acids can also be self‐activated by N‐phosphorylation. This information not only provides some interesting clues for understanding the active role of the phosphoryl group in living systems, but also shows that the origin of life might be attributed to the chemical evolution of N‐phosphoryl amino acids.

An Isotope (18O, 15N, and 2H) Technique to Investigate the Metal Ion Interactions Between the Phosphoryl Group and Amino Acid Side Chains by Electrospray Ionization Mass Spectrometry

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope ((18)O, (15)N, and (2)H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.

From sequence to structural analysis in protein phosphorylation motifs

Phosphorylation is the most widely studied post-translational modification occurring in cells. While mass spectrometry-based proteomics experiments are uncovering thousands of novel in vivo phosphorylation sites, the identification of kinase specificity rules still remains a relatively slow and often inefficacious task. In the last twenty years, many efforts have being devoted to the experimental and computational identification of sequence and structural motifs encoding kinase-substrate interaction key residues and the phosphorylated amino acid itself. In this review, we retrace the road to the discovery of phosphorylation sequence motifs, examine the progresses achieved in the detection of three-dimensional motifs and discuss their importance in the understanding of regulation and de-regulation of many cellular processes.

Contribution of Alanine-76 and Serine Phosphorylation inα-Synuclein Membrane Association and Aggregation in Yeasts

In Parkinson's disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synuclein's plasma membrane and endomembrane association.

Optimization of Peptide Density on Microarray Surface for Quantitative Phosphoproteomics

Procedures to determine the density of peptides immobilized on a glass surface for the quantitative detection of phosphorylated peptides for phosphoproteomic applications of peptide microarrays are described. Two kinds of representative fluorescent probe molecules, anti-phosphotyrosine antibody (AB) and phos-tag (PT), were examined to compare their ability for the quantitative detection of phosphorylated peptides. PT is a metal complex with a binding specificity to phosphorylated amino acids, and is much smaller in size than AB. Thus, PT is quantitatively bound to the phosphorylated peptides, even at a high immobilization density without steric hindrance, making them highly suited for future microarrays requiring smaller sized peptide spots for much higher throughput.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor‐mediated signalling in cooperation with Nod factor receptor 5

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.

Analysis of earthworm Eisenia fetida proteomes during cadmium exposure: An ecotoxicoproteomics approach

Identification of differential proteomic responses to cadmium (Cd) would provide a means for better understanding the survival mechanisms of the earthworm, Eisenia fetida, living in a Cd-polluted environment, and the stress responses can be assessed by ecotoxiptoromics approaches. Extracts of whole earthworm collected at days 7, 14, 21, and 28 after Cd exposure were analyzed by 2-DE and quantitative image analysis. In total, 143 proteins demonstrated significant regulation in at least at one time point, and 56 proteins were identified by MALDI-TOF/TOF-MS and database searching. Compared with control samples, 28 protein spots were up-regulated and 28 were down-regulated during at least one time point. The identified proteins, including chaperonine protein HSP60, caspase-8, calcium ion-binding protein, zinc ion-binding protein, actin-binding protein, proteolysis, fibrinolytic protease, glutamate dehydrogenase, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor 1, manganous superoxide dismutase, extracellular globin-4, lysenin, intermediate filament protein, and tubulin, are involved in several processes, including transcription, translation, the tricarboxylic acid cycle, the cellular amino acid metabolic process, protein amino acid phosphorylation, glycolysis, and the glucose metabolic process. Thus, our study provides a functional profile of the Cd-responsive proteins in earthworms.

Preparation and adsorption properties of phospho‐L‐glutamic acid on hydroxyapatite

AbstractPhospho‐L‐glutamic acid was successfully prepared by the phosphorylation of glutamic acid, and its adsorption on synthetic hydroxyapatite (HAP) was studied together with glutamic. The adsorption behaviors of both adsorbates were adequately described by a Langmuirian model. From the comparison between two different adsorbates, the results of the investigation indicated that the phosphate group in phospho‐L‐glutamic acid can greatly enhanced the adsorption affinity for HAP, the improvement of which was achieved through replacing the same group on the surface of HAP and interacting with the surface calcium ion of HAP by electrostatic attraction. The results obtained laid the solid foundation for further research on the regulating function of phosphorylated amino acids with hydroxyapatite biological composites. Copyright © 2010 John Wiley & Sons, Ltd.

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Hyperphosphorylation by Cyclin B/CDK1 in Mitosis Resets CUX1 DNA Binding Clock at Each Cell Cycle*

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).

Esterification of an Unnatural Amino Acid Structurally Deviating from Canonical Amino Acids Promotes Its Uptake and Incorporation into Proteins in Mammalian Cells

Genetically encoded unnatural amino acids (UAAs) enable novel chemical and physical properties to be selectively introduced into proteins directly in live cells, which provides great potential for addressing biological questions at the molecular and cellular level in native settings.[1-3] UAAs have been genetically incorporated into proteins in mammalian cells using orthogonal tRNA-codon-synthetase sets,[4-6] yet the current low incorporation efficiency hinders their effective application. Efforts to improve efficiency have focused on optimizing the expression and activity of the orthogonal tRNA and synthetase,[5-7] whereas the bioavailability of the UAA inside mammalian cells, a prerequisite for incorporation, has not been addressed. In addition, there are many UAAs that have not been genetically incorporated into proteins successfully, such as glycosylated and phosphorylated amino acids. These amino acids can be invaluable for studying the contribution of posttranslational modifications to protein function and the role of a target protein in cellular signal transduction. Among many reasons, the inability of the UAA to enter cells prevents evolving a mutant synthetase specific for the UAA using cell-based selections or screens.[2] Here we show that an UAA structurally deviating from the canonical amino acids in side chain could not be efficiently transported into mammalian cells, but masking the carboxyl group of the UAA as an ester greatly increased the rate of cellular uptake and intracellular concentration of the UAA. This resulted in a significant increase in the incorporation of this UAA into proteins in mammalian cells. Among three esters tested, acetoxymethyl ester (AME) yielded the highest UAA incorporation efficiency with a concomitant reduction of the UAA required in the growth media.

Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

With myocardial infarction (MI), cardiac troponin is released from the heart into circulation, where it can be detected with immunoassays independently quantifying cardiac troponin I (cTnI) or cTnT. There is, however, no single immunoassay that sequentially probes the posttranslational modification status of cTnI or directly characterizes whether circulating cTnI is bound to cTnC and/or cTnT. Here we describe the development of a qualitative immunoassay to directly probe the primary and ternary structure of circulating cTnI through diffractive optics technology (dotLab System, Axela). Anti-cTnI antibody 8I-7 was immobilized on a patterned sensor to capture cTnI. One or more detector antibodies were sequentially introduced to probe for amino acid sequence integrity or phosphorylation status of cTnI, or its association with cTnC and/or cTnT. Respective immunocaptures were recorded as real-time diffractive intensities (DIs), and the DI differences were analyzed. Each immunodetection was independent of the others but was done in a single sequential assay. This diffraction-based immunoassay successfully characterized cTnI. The unamplified assay determined whether cTnI was degraded at N-terminus and/or C-terminus or phosphorylated. Sequential application of multiple detector antibodies without an antibody-stripping step enables real-time interrogation of 5 different epitopes of cTnI, or direct detection of the cTn complex (cTnI-cTnC-cTnT) in a single sequential assay. Finally, this assay was optimized with amplification to directly detect circulating cTnI bound to cTnC and cTnT in serum from an MI patient. The dot Immunoassay is the first qualitative sequential immunoassay to address the direct interactions of the troponin subunits and various modified forms of cTnI.

Amino Acid Homochirality may be Linked to the Origin of Phosphate-Based Life

Phosphorylation has to have been one of the key events in prebiotic evolution on earth. In this article, the emergence of phosphoryl amino acid 5'-nucleosides having a P-N bond is described as a model of the origin of amino acid homochirality and Genetic Code. It is proposed that the intramolecular interaction between the nucleotide base and the amino acid side-chain influences the stability of particular amino acid 5'-nucleotides, and the interaction also selects for the chirality of amino acids. The differences between L: - and D: -conformation energies (DeltaE (conf)) are evaluated by DFT methods at the B3LYP/6-31G(d) level. Although, as expected, these DeltaE (conf) values are not large, they do give differences in energy that can distinguish the chirality of amino acids. Based on our calculations, the chiral selection of the earliest amino acids for L: -enantiomers seems to be determined by a clear stereochemical/physicochemical relationship. As later amino acids developed from the earliest amino acids, we deduce that the chirality of these late amino acids was inherited from that of the early amino acids. This idea reaches far back into evolution, and we hope that it will guide further experiments in this area.

Constitutive activation of the mitogen‐activated protein kinase pathway impairs vitamin D signaling in human prostate epithelial cells

We studied the effect of prolonged activation of mitogen-activated protein kinase (MAPK) signaling on 1,25 dihydroxyvitamin D (1,25(OH)(2)D(3)) action in the immortalized human prostate epithelial cell line RWPE1 and its Ki-Ras transformed clone RWPE2. 1,25(OH)(2)D(3)-treatment caused growth arrest and induced gene expression in both cell lines but the response was blunted in RWPE2 cells. Vitamin D receptor (VDR) levels were lower in RWPE2 cells but VDR over-expression did not increase vitamin-D-mediated gene transcription in either cell line. In contrast, MAPK inhibition restored normal vitamin D transcriptional responses in RWPE2 cells and MAPK activation with constitutively active MEK1R4F reduced vitamin-D-regulated transcription in RWPE1 cells. 1,25(OH)(2)D(3)-mediated transcription depends upon the VDR and its heterodimeric partner the retinoid X receptor (RXR) so we studied whether changes in the VDR-RXR transcription complex occur in response to MAPK activation. Mutation of putative phosphorylation sites in the activation function 1 (AF-1) domain (S32A, T82A) of RXRalpha restored 1,25(OH)(2)D(3)-mediated transactivation in RWPE2 cells. Mammalian two-hybrid and co-immunoprecipitation assays revealed a vitamin-D-independent interaction between steroid receptor co-activator-1 (SRC-1) and RXRalpha that was reduced by MAPK activation and was restored in RWPE2 cells by mutating S32 and T82 in the RXRalpha AF-1 domain. Our data show that a common contributor to cancer development, prolonged activation of MAPK signaling, impairs 1,25(OH)(2)D(3)-mediated transcription in prostate epithelial cells. This is due in part to the phosphorylation of critical amino acids in the RXRalpha AF-1 domain and impaired co-activator recruitment.