PIM1 phosphorylation of the androgen receptor and 14-3-3 ζ coordinates their interaction and chromatin occupancy to regulate gene transcription in prostate cancer
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE181226 | GEO | 2021/11/03
REPOSITORIES: GEO
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Project description:PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.
| S-EPMC8546101 | biostudies-literature
Project description:PIM1 phosphorylation of the androgen receptor and 14-3-3 ζ coordinates their interaction and chromatin occupancy to regulate gene transcription in prostate cancer
| PRJNA751077 | ENA
Project description:We previously identified 14-3-3 ζ S64 as a substrate of PIM1 in an in vivo chemical genetic screen. To verify this phosphorylation event and determine whether S64 is the predominant phosphorylation site, we performed in vitro phosphorylation of 14-3-3 ζ by PIM1.
2021-11-25 | PXD023623 | Pride
Project description:Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.
| S-EPMC3527659 | biostudies-literature
Project description:We used ChIP-seq to identify chromatin binding sites for AR, 14-3-3 ζ, and pS213 AR in the absence and presence of PIM1 over-expression. We find that, as expected, pS213 AR occupancy almost completely overlaps with total AR occupancy, and increases in the presence of PIM1 over-expression. Additionally, the majority of pS213 AR sites are co-occupied by 14-3-3 ζ. 14-3-3 ζ is a known AR co-activator, but this study definitively shows its co-occupancy on chromatin with AR and pS213 AR.
2021-11-03 | GSE181224 | GEO
Project description:Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3?-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3?. Interestingly, OGT activity is regulated by GSK3?; hence, OGT and GSK3? exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.
| S-EPMC3597447 | biostudies-literature
Project description:14-3-3 ζ is an androgen receptor (AR) co-activator. To further understand how 14-3-3 ζ may be modulating AR activity, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins for analysis of chromatin complexes (RIME) on AR and 14-3-3 ζ in LNCaP prostate cancer cells in the presence of PIM1. This enabled us to observe what other proteins associate with both 14-3-3 ζ and AR on chromatin. Notably, several of the proteins we identified as associated with both AR and 14-3-3 ζ are known AR co-regulators. We hypothesized that to co-regulate AR, 14-3-3 ζ may be recruiting or altering interactions between AR and AR co-regulators.
2021-11-25 | PXD023634 | Pride
Project description:LRRK2 mutations are among the most common genetic causes for Parkinson's disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3θ isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3θ phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3θ mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3θ mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3θ mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3θ phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3θ and phosphorylated T2524 LRRK2 was important for 14-3-3θ's ability to regulate kinase activity, as wildtype and S232A 14-3-3θ failed to reduce the kinase activity of G2019S/T2524A LRRK2. Molecular modeling showed that 14-3-3θ phosphorylation causes a partial rearrangement of its canonical binding pocket, thus affecting the interaction between 14-3-3θ and the C-terminus of LRRK2. We conclude that 14-3-3θ phosphorylation destabilizes the interaction of 14-3-3θ with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity.
| S-EPMC10312468 | biostudies-literature
Project description:Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3?, ?, and ? isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3? and ? binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3? increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3? knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3? interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3? interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.
| S-EPMC3302758 | biostudies-literature
Project description:PURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action. Experimental Design and RESULTS: Androgen receptor and 14-3-3eta colocalized in COS cell nuclei with and without androgen, and 14-3-3eta promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3eta interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3eta at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor-dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3eta. In the CWR22 prostate cancer xenograft model, 14-3-3eta expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3eta and androgen receptor actions. 14-3-3eta mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3eta levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3eta localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. CONCLUSION: 14-3-3eta enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571-81).
| S-EPMC2795092 | biostudies-literature