Quantification of HER2 in COS7 cells using quantum weak measurement.

A Mach-Zehnder interferometer system based on weak measurement was set up to determinate the concentration variation of molecule by measuring the phase difference change between the two optical paths. The spectrum of the light was recorded to monitor the concentration of trastuzumab (Herceptin), which is a humanised monoclonal antibody, targeted to human epidermal growth factor receptor 2 (HER2). The trastuzumab targeting to HER2 was real-time detected and continuously monitored, the HER2 numbers of COS7 cells on a coverslip was determined at pico-molar level. Our weak measurement enabled method proposes an alternative approach for the concentration detection of molecules, providing a promising functional tool for the quantification of HER2 in cancer cells, possibly promoting fields such as the diagnosis and treatment of cancer.

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography.

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.

Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.

AGPAT (1-acyl-sn-glycerol 3-phosphate acyltransferase) exists in at least five isoforms in humans, termed as AGPAT1, AGPAT2, AGPAT3, AGPAT4 and AGPAT5. Although they catalyse the same biochemical reaction, their relative function, tissue expression and regulation are poorly understood. Linkage studies in humans have revealed that AGPAT2 contributes to glycerolipid synthesis and plays an important role in regulating lipid metabolism. We report the molecular cloning, tissue distribution, and enzyme characterization of mAGPATs (murine AGPATs) and regulation of cardiac mAGPATs by PPARalpha (peroxisome-proliferator-activated receptor alpha). mAGPATs demonstrated differential tissue expression profiles: mAGPAT1 and mAGPAT3 were ubiquitously expressed in most tissues, whereas mAGPAT2, mAGPAT4 and mAGPAT5 were expressed in a tissue-specific manner. mAGPAT2 expressed in in vitro transcription and translation reactions and in transfected COS-1 cells exhibited specificity for 1-acyl-sn-glycerol 3-phosphate. When amino acid sequences of five mAGPATs were compared, three highly conserved motifs were identified, including one novel motif/pattern KX2LX6GX12R. Cardiac mAGPAT activities were 25% lower (P<0.05) in PPARalpha null mice compared with wild-type. In addition, cardiac mAGPAT activities were 50% lower (P<0.05) in PPARalpha null mice fed clofibrate compared with clofibrate fed wild-type animals. This modulation of AGPAT activity was accompanied by significant enhancement/reduction of the mRNA levels of mAGPAT3/mAGPAT2 respectively. Finally, mRNA expression of cardiac mAGPAT3 appeared to be regulated by PPARalpha activation. We conclude that cardiac mAGPAT activity may be regulated by both the composition of mAGPAT isoforms and the levels of each isoform.

Low-density lipoprotein from apolipoprotein E-deficient mice induces macrophage lipid accumulation in a CD36 and scavenger receptor class A-dependent manner.

OBJECTIVE: To investigate the potential of circulating low-density lipoprotein (LDL), isolated from apolipoprotein E (apoE)-deficient mice (E-/-LDL) and from LDL receptor-deficient mice (Lr-/-LDL), to induce foam cell formation. METHODS AND RESULTS: Binding studies using COS-7 cells overexpressing CD36, J774 cells, and mouse peritoneal macrophages (MPMs) unexpectedly showed for the first time that E-/-LDL, which is enriched in cholesterol, is a high-affinity ligand for CD36 and exhibited greater macrophage uptake than Lr-/-LDL or normal LDL. Minimal copper-mediated oxidization of Lr-/-LDL or C57LDL in vitro resulted in increased ligand internalization, although cell uptake of these oxidized LDLs was lower than that of E-/-LDL, even at oxidation levels similar to that found in E-/-LDL. Treatment of MPMs with E-/-LDL and Lr-/-LDL (to a 2- to 3-fold lesser extent), but not normal LDL, resulted in significant cellular cholesteryl ester accumulation and foam cell formation. Experiments using MPMs lacking CD36, scavenger receptor class A (SR-A), or both, indicated a major contribution of CD36 ( approximately 50%), and to a lesser extent, SR-A (24% to 30%), to E-/-LDL uptake. CONCLUSIONS: Because of its increased state of oxidation and high cholesterol content, LDL in apoE-deficient mice acts in a proatherogenic manner, without requiring further modification in the vascular wall, to induce foam cell formation through its uptake by scavenger receptors.

Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.

Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein.

OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface. METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal. Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.

BACKGROUND: C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results. METHODS AND RESULTS: To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude. CONCLUSIONS: Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

Identification and regulation of Sprouty1, a negative inhibitor of the ERK cascade, in the human heart.

We screened a compendium of gene profiles from 19 paired human heart samples harvested at the time of implant and explant of a left ventricular assist device (LVAD) for novel genes regulating the Ras/MEK/ERK cascade. From this analysis we identified Sprouty1, an evolutionally conserved gene that acts as an intrinsic inhibitor of the Ras/MEK/ERK pathway. Sprouty1 mRNA and protein were significantly upregulated in the heart in response to mechanical unloading with a LVAD. The upregulation of Sprouty1 in the heart following mechanical unloading was accompanied by a significant decrease in phosphorylated ERK1/2. Gain of function experiments demonstrated that upregulation of Sprouty1 in isolated cardiac myocytes led to a significant decrease and altered kinetics of ERK1/2 phosphorylation. Immunohistochemistry of human hearts revealed that Sprouty1 was also expressed in the microvasculature. Upregulation of Sprouty1 in endothelial cells led to a significant decrease in VEGF-induced endothelial cell proliferation. To our knowledge, these findings are the first to define Sprouty expression in the heart and suggest that Sprouty1 may serve as an intrinsic mediator governing ventricular remodeling through a coordinated coupling of both myocyte and vascular alterations in response to mechanical load.

Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor alpha by AMP-activated protein kinase.

AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARalpha (peroxisome proliferator-activated receptor alpha) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARalpha-mediated transcription is shown here to be co-activated by the alpha subunit of AMPK, as well as by kinase-deficient (Thr172Ala) and kinase-less (Asp157Ala, Asp139Ala) mutants of AMPKalpha. The Ser452Ala mutant of mPPARalpha mutated in its putative consensus AMPKalpha phosphorylation site is similarly co-activated by AMPKalpha. AMPKalpha or its kinase-less mutants bind to PPARalpha; binding is increased by MgATP, to a lesser extent by MgADP, but not at all by AMP or ZMP [AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) monophosphate]. ATP-activated binding of AMPKalpha to PPARalpha is mediated primarily by the C-terminal regulatory domain of AMPKalpha. PPARalpha co-activation by AMPKalpha may, however, require its secondary interaction with the N-terminal catalytic domain of AMPKalpha, independently of its kinase activity. While AMPK catalytic activity is activated by AICAR, PPARalpha co-activation and PPARalpha-controlled transcription are robustly inhibited by AICAR, with concomitant translocation of nuclear AMPKalpha or its kinase-less mutants to the cytosol. In conclusion, AMPKalpha, independently of its kinase activity, co-activates PPARalpha both in primary rat hepatocytes and in PPARalpha-transfected cells. The kinase and transcriptional co-activation modes of AMPKalpha are both regulated by the cellular ATP/AMP ratio. Co-activation of PPARalpha by AMPKalpha may transcriptionally complement AMPK in maintaining cellular ATP status.

Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4).

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50-80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469-->Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.

Synergistic role of specificity proteins and upstream stimulatory factor 1 in transactivation of the mouse carboxylesterase 2/microsomal acylcarnitine hydrolase gene promoter.

Mouse carboxylesterase 2 (mCES2), a microsomal acylcarnitine hydrolase, is thought to play some important roles in fatty acid (ester) metabolism, and it is therefore thought that the level of transcription of the mCES2 gene is under tight control. Examination of the tissue expression profiles revealed that mCES2 is expressed in the liver, kidney, small intestine, brain, thymus, lung, adipose tissue and testis. When the mCES2 promoter was cloned and characterized, it was revealed that Sp1 (specificity protein 1) and Sp3 could bind to a GC box, that USF (upstream stimulatory factor) 1 could bind to an E (enhancer) box, and that Sp1 could bind to an NFkappaB (nuclear factor kappaB) element in the mCES2 promoter. Co-transfection assays showed that all of these transcription factors contributed synergistically to transactivation of the mCES2 promoter. Taken together, our results indicate that Sp1, Sp3 and USF1 are indispensable factors for transactivation of the mCES2 gene promoter. To our knowledge, this is the first study in which transcription factors that interact with a CES2 family gene have been identified. The results of the present study have provided some clues for understanding the molecular mechanisms regulating mCES2 gene expression, and should be useful for studies aimed at elucidation of physiological functions of mCES2.

Two new FUT2 (fucosyltransferase 2 gene) missense polymorphisms, 739G-->A and 839T-->C, are partly responsible for non-secretor status in a Caucasian population from Northern Portugal.

Secretor status is defined by the expression of H type 1 antigen on gastric surface epithelium and external secretions. The H type 1 structure, and other fucosylated carbohydrates (Le(a), sialyl-Le(a), Le(b), Le(x), sialyl-Le(x) and Le(y)), can serve as ligands for several pathogens, including Helicobacter pylori, and are cancer-associated antigens. Secretor individuals are more susceptible to some bacterial and viral infections of the genito-urinary and digestive tracts. The aim of the present study was to examine FUT2 (fucosyltransferase 2 gene) polymorphisms in a Caucasian population of non-secretor individuals (n=36) from northern Portugal and to evaluate the activity of the mutant FUT2 enzymes. The secretor status was determined by UEAI [Ulex europaeus (gorse) lectin] histochemistry in gastric mucosa, and FUT2 polymorphisms were studied by restriction-fragment-length polymorphism and direct sequencing. The majority of non-secretors (88.9%) were homozygous for 428G-->A polymorphism; 5.6% were homozygous for 571C-->T and 5.6% were homozygous for two new missense polymorphisms, 739G-->A (2.8%) and 839T-->C (2.8%). By kinetic studies it was demonstrated that the two new FUT2 mutants (739G-->A and 839T-->C) are almost inactive and are responsible for some non-secretor cases.

Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter.

Glutamine transport into the human hepatoma cell line HepG2 is catalysed primarily by an ASCT2-type transporter identical in sequence with that cloned previously from JAR cells. An antibody raised against the C-terminus of the ASCT2 protein was shown to recognize ASCT2 on Western blots. Using this antibody, it was found that variation in cell growth rate did not affect ASCT2 expression, but both growth rate and ASCT2 expression were significantly reduced by glutamine deprivation. Expression of a number of other proteins was shown to be unaffected under these conditions. The sequence of the 5'-flanking region of the ASCT2 gene was derived from the human genome database. A 907 bp fragment of this sequence was directionally ligated into a luciferase reporter vector and was shown to exhibit promoter activity when transfected into HepG2 cells. Promoter activity was greatly reduced when transfection was performed in glutamine-free medium and was restored when glutamine was added post-transfection. The absence of other essential amino acids did not affect promoter activity, and glutamine deprivation did not affect the MCT1 (monocarboxylate transporter 1) promoter. These results indicate that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability.

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence. With spacers of < or =12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP-FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.

Delta-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) interacts with and activates sphingosine kinase 1.

Sphingosine kinase (SPHK) is a key enzyme catalysing the formation of sphingosine 1-phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events acting through intracellular, as well as extracellular, mechanisms. However, the molecular mechanism of intracellular actions of SPP remains unclear. Here, we have identified delta-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) as a potential binding partner for SPHK1 by yeast two-hybrid screening. From co-immunoprecipitation analyses, the C-terminal portion of delta-catenin/NPRAP containing the seventh to tenth armadillo repeats was found to be required for interaction with SPHK1. Endogenous delta-catenin/NPRAP was co-localized with endogenous SPHK1 and transfected delta-catenin/NPRAP was co-localized with transfected SPHK1 in dissociated rat hippocampal neurons. MDCK (Madin-Darby canine kidney) cells stably expressing delta-catenin/NPRAP contained elevated levels of intracellular SPP. In a purified system delta-catenin/NPRAP stimulated SPHK1 in a dose-dependent manner. Furthermore, delta-catenin/NPRAP-induced increased cell motility in MDCK cells was completely inhibited by dimethylsphingosine, a specific inhibitor of SPHK1. These results strongly suggest that at least some of delta-catenin/NPRAP functions, including increased cell motility, are mediated by an SPHK-SPP signalling pathway.

Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells.

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.

The Icelandic founder mutation BRCA2 999del5: analysis of expression.

INTRODUCTION: A founder mutation in the BRCA2 gene (BRCA2 999del5) accounts for 7-8% of female breast cancers and for 40% of male breast cancers in Iceland. If expressed, the mutant gene would encode a protein consisting of the first 256 amino acids of the BRCA2 protein. The purpose of this study was to determine whether this mutant protein is produced in heterozygous individuals and, if so, what might be the functional consequences of mutant protein production. METHODS: The presence of BRCA2 999del5 transcripts in fibroblasts from heterozygous individuals was assayed by cDNA synthesis and sequencing. The potential protein-coding portion of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His vector and expressed in COS7 cells. The presence of the mutant protein in cell lysates from heterozygous fibroblasts and from COS7 cells was tested by a number of methods including immunoprecipitation, affinity purification with nickel-coated agarose beads, Western blotting and ELISA, using antibodies to the N-terminal end of BRCA2, antiserum specific for the 16 nonrelevant amino acids at the carboxyl end and antibodies to fusion partners of recombinant proteins. RESULTS: The frequency of the BRCA2 999del5 transcript in heterozygous fibroblasts was about one-fifth of the wild-type transcript; however, no mutant protein could be detected. Overexpression of BRCA2 999del5 mRNA in COS7 cells failed to produce a mutant protein unless degradation by proteasomes was blocked. CONCLUSION: Our results show that the protein product of BRCA2 999del5 is extremely unstable. Therefore, an increase in breast cancer risk in BRCA2 999del5 carriers is due to haploinsufficiency at the BRCA2 locus.

p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cdelta.

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.

Analysis of two CBP (cAMP-response-element-binding protein-binding protein) interacting sites in GRIP1 (glucocorticoid-receptor-interacting protein), and their importance for the function of GRIP1.

The p160 co-activators, SRC1 (steroid receptor co-activator 1), GRIP1 (glucocorticoid-receptor-interacting protein 1) and ACTR (activator for thyroid hormone and retinoid receptors), have two ADs (activation domains), AD1 and AD2. AD1 is a binding site for the related co-activators, CBP (cAMP-response-element-binding protein-binding protein) and p300, whereas AD2 binds to another co-activator, co-activator-associated arginine methyltransferase 1 (CARM1). Here, we identified two CBP-interacting sites [amino acids 1075-1083 (site I) and 1095-1106 (site II)] in a so-called CBP-dependent transactivation domain (AD1; amino acids 1057-1109) of GRIP1. Site I was the major site for CBP-dependent AD1 transactivation activity of GRIP1 whereas, following the deletion of site II, full or partial transactivation activity was retained without the recruitment of CBP in yeast, HeLa, human embryonic kidney 293 and CV-1 cells. GRIP1 (with a deletion of site II) expressed stronger co-activator activity than that of wild-type GRIP1 in the TR (thyroid receptor) and the AR (androgen receptor), but not the ER (oestrogen receptor), systems in HeLa cells. We also demonstrated that these CBP-binding sites of GRIP1 are not the only functional domains for its AD1 function in TR, AR and ER systems in HeLa cells by the exogenous overexpression of one E1A mutant, which led to a lack of CBP-binding ability. Our results suggest that these two CBP-interacting sites in the GRIP AD1 domain not only determine its AD1 activity, but are also involved in its co-activator functions in some nuclear receptors.