ERK2 phosphorylation of EBNA1 serine 383 residue is important for EBNA1-dependent transactivation.
Ontology highlight
ABSTRACT:
SUBMITTER: Noh KW
PROVIDER: S-EPMC5041921 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.
| S-EPMC3067954 | biostudies-literature
Project description:Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S(136), that undergoes increased phosphorylation upon elevation of intracellular Ca(2+) concentration. A phosphospecific antibody (pS(136)) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84, and HEK293 cells. By using D52 as a substrate, a protein kinase with a molecular weight (M(r)) of approximately 50 kDa was identified with "in gel" assays. This kinase comigrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2)alpha cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT(286/287)) antibody when activated. Carbachol-stimulated phosphorylation of S(136) was inhibited by the CAMK2 inhibitor KN93 (IC(50) 38 microM) and by the calmodulin antagonist W7 (IC(50) 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2delta6, which has the same domain structure and M(r) as CAM2alpha, was identified in gastric mucosa by RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK2delta6 in the mediation of D52 phosphorylation.
| S-EPMC2604800 | biostudies-literature
Project description:Biomarkers for predicting prognosis are critical to treating colorectal cancer (CRC) patients. We found that CSN6, a subunit of COP9 signalosome, is overexpressed in CRC samples and that CSN6 overexpression is correlated with poor patient survival. Mechanistic studies revealed that CSN6 is deregulated by epidermal growth factor receptor (EGFR) signaling, in which ERK2 binds directly to CSN6 Leu163/Val165 and phosphorylates CSN6 at Ser148. Furthermore, CSN6 regulated β-Trcp and stabilized β-catenin expression by blocking the ubiquitin-proteasome pathway, thereby promoting CRC development. High CSN6 expression was positively correlated with ERK2 activation and β-catenin overexpression in CRC samples, and inhibiting CSN6 stability with cetuximab reduced colon cancer growth. Taken together, our study's findings indicate that the deregulation of β-catenin by ERK2-activated CSN6 is important for CRC development.
| S-EPMC4560098 | biostudies-literature
Project description:Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage-specific event that is downstream of ATR and is functionally important in the FA pathway.
| S-EPMC2652366 | biostudies-literature
Project description:ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.
| S-EPMC4163909 | biostudies-literature
Project description:Human BCL-2-associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.
| S-EPMC6217419 | biostudies-literature
Project description:NRIF3 is an estrogen-inducible nuclear receptor coregulator that stimulates estrogen receptor-alpha (ERalpha) transactivation functions and associates with the endogenous ER and its target gene promoter. p21-activated protein kinase 1 (Pak1) phosphorylates ERalpha at Ser305 and this modification is important in ERalpha transactivation function. Although ERalpha transactivation functions are regulated by co-activator activity of NRIF3, it remains unclear whether Pak1 could impact ER functions via a posttranslational modification of NRIF3. Here, we report that Pak1 phosphorylates NRIF3 at Serine28 and that NRIF3 binds to Pak1 in vitro and in vivo. We found that NRIF3 phosphorylation, co-activator activity and association with ERalpha increased following Pak1 phosphorylation of NRIF3's Ser28 and that activated ERalpha-Ser305 and NRIF3-Ser28 cooperatively support transactivation of ERalpha. NRIF3 expression increased significantly in cells with inducible Pak1 expression. We found that NRIF3 and ERalpha interaction, subcellular localization and ERalpha transactivation activity all increased in cells expressing the Pak1 phosphorylation-mimicking mutant NRIF3-Ser28Glu. Consistently, the NRIF3-Ser28Glu mutant exhibited an enhanced recruitment to the endogenous ER target genes and increased expression following estrogen stimulation. Finally, breast cancer cells with stable overexpression of NRIF3 showed increased proliferation and enhanced anchorage-independent growth. These findings suggest that NRIF3-Ser28 is a physiologic target of Pak1 signaling and contributes to the enhanced NRIF3 co-activator activity, leading to coordinated potentiation of ERalpha transactivation, its target gene expression and estrogen responsiveness of breast cancer cells.
| S-EPMC3621709 | biostudies-literature
Project description:Although STAT1 tyrosine-701 phosphorylation (designated STAT1-pY701) is indispensable for STAT1 function, the requirement for STAT1 serine-727 phosphorylation (designated STAT1-pS727) during systemic autoimmune and antipathogen responses remains unclear. Using autoimmune-prone B6.Sle1b mice expressing a STAT1-S727A mutant in which serine is replaced by alanine, we report in this study that STAT1-pS727 promotes autoimmune Ab-forming cell (AFC) and germinal center (GC) responses, driving autoantibody production and systemic lupus erythematosus (SLE) development. In contrast, STAT1-pS727 is not required for GC, T follicular helper cell (Tfh), and Ab responses to various foreign Ags, including pathogens. STAT1-pS727 is also not required for gut microbiota and dietary Ag-driven GC and Tfh responses in B6.Sle1b mice. By generating B cell-specific bone marrow chimeras, we demonstrate that STAT1-pS727 plays an important B cell-intrinsic role in promoting autoimmune AFC, GC, and Tfh responses, leading to SLE-associated autoantibody production. Our analysis of the TLR7-accelerated B6.Sle1b.Yaa SLE disease model expressing a STAT1-S727A mutant reveals STAT1-pS727-mediated regulation of autoimmune AFC and GC responses and lupus nephritis development. Together, we identify previously unrecognized differential regulation of systemic autoimmune and antipathogen responses by STAT1-pS727. Our data implicate STAT1-pS727 as a therapeutic target for SLE without overtly affecting STAT1-mediated protection against pathogenic infections.
| S-EPMC9305983 | biostudies-literature
Project description:The transcription factor Stat1 plays an essential role in responses to interferons (IFNs). Activation of Stat1 is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity and biological function Stat1 must also be phosphorylated on S727. The molecular mechanisms underlying the IFN-induced S727 phosphorylation are incompletely understood. Here, we show that both Stat1 Y701 phosphorylation and nuclear translocation are required for IFN-induced S727 phosphorylation. We further show that Stat1 mutants lacking the ability to stably associate with chromatin are poorly serine-phosphorylated in response to IFN-gamma. The S727 phosphorylation of these mutants is restored on IFN-beta treatment that induces the formation of the ISGF3 complex (Stat1/Stat2/Irf9) where Irf9 represents the main DNA binding subunit. These findings indicate that Stat1 needs to be assembled into chromatin-associated transcriptional complexes to become S727-phosphorylated and fully biologically active in response to IFNs. This control mechanism, which may be used by other Stat proteins as well, restricts the final activation step to the chromatin-tethered transcription factor.
| S-EPMC2435588 | biostudies-literature
Project description:Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 A distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.
| S-EPMC1303804 | biostudies-literature