Receptor mechanisms of rapid extranuclear signalling initiated by steroid hormones

2004 ◽  
Vol 40 ◽  
pp. 105-120 ◽  
Author(s):  
Viroj Boonyaratanakornkit ◽  
Dean P Edwards

In addition to their role as direct regulators of gene transcription mediated by classical nuclear hormone receptors, steroid hormones have also been described to exert rapid effects on intracellular signalling pathways independent of gene transcription. This chapter focuses on recent advances in our understanding of the receptors and mechanisms that mediate these rapid signalling actions of oestrogens and progesterone. Increasing evidence suggests that at least some of these rapid actions are mediated by a subpopulation of the classical nuclear oestrogen receptor (ER) and progesterone receptor (PR) that localize to the cytoplasm or associate with the plasma membrane. Human PR has been shown to mediate rapid progestin activation of the Src/Ras/Raf/mitogen-activated protein kinase signalling pathway in mammalian cells by a direct interaction with the Src homology 3 domain of Src tyrosine kinases through a Pro-Xaa-Xaa-Pro-Xaa-Arg motif located in the N-terminal domain of the receptor. Moreover, this is an extranuclear action of PR that is separable from its direct transcriptional activity. Additionally, a novel membrane protein unrelated to nuclear PR was recently identified that has properties of a G-protein-coupled receptor for progesterone and has been shown to be involved in mediating the extranuclear signalling actions of progesterone that promotes oocyte maturation in fish. The role of this membrane PR (mPR) in mammalian cells is less clear and the relationship of the membrane and classical nuclear PR in mediating rapid non-transcriptional signalling of progestins has not been explored. To date, a novel membrane ER unrelated to classical nuclear receptors has not been cloned and characterized, and many of the known rapid extranuclear signalling actions of oestrogen appear also to be mediated by a subpopulation of nuclear ER, or a closely related receptor. A novel protein termed modulator of non-genomic activity of ER (MNAR) has been identified that acts as an adaptor between ER and Src, and thus provides a mechanisms for coupling of oestrogen and ER with rapid oestrogen-induced activation of Src and the downstream mitogen-activated protein kinase signalling cascade. The physiological relevance of rapid extranuclear signalling by the classical ER has been provided by experiments showing that these actions contribute to the anti-apoptotic effect of oestrogen in bone in vivo and to the rapid effects of oestrogen on vasodilation and protection of endothelial cells against injury.

2011 ◽  
Vol 438 (3) ◽  
pp. 495-503 ◽  
Author(s):  
Ratnesh K. Srivastav ◽  
Susan Schwede ◽  
Malte Klaus ◽  
Jessica Schwermann ◽  
Matthias Gaestel ◽  
...  

Protein–protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein–protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein–protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α–Edr2 interaction was verified by co-localization analysis. Functionally, this p38α–Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α–MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)–MK5 and of the p38α–p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.


2004 ◽  
Vol 24 (24) ◽  
pp. 10954-10964 ◽  
Author(s):  
Charles E. Foulds ◽  
Mary L. Nelson ◽  
Adam G. Blaszczak ◽  
Barbara J. Graves

ABSTRACT Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.


2005 ◽  
Vol 25 (24) ◽  
pp. 10695-10710 ◽  
Author(s):  
Pradeep K. Pandey ◽  
T. S. Udayakumar ◽  
Xinjie Lin ◽  
Dipali Sharma ◽  
Paul S. Shapiro ◽  
...  

ABSTRACT The TRAP/Mediator coactivator complex serves as a molecular bridge between gene-specific activators and RNA polymerase II. TRAP220/Med1 is a key component of TRAP/Mediator that targets the complex to nuclear hormone receptors and other types of activators. We show here that human TRAP220/Med1 is a specific substrate for extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. We demonstrate that ERK phosphorylates TRAP220/Med1 in vivo at two specific sites: threonine 1032 and threonine 1457. Importantly, we found that ERK phosphorylation significantly increases the stability and half-life of TRAP220/Med1 in vivo and correlates with increased thyroid hormone receptor-dependent transcription. Furthermore, ERK phosphorylates TRAP220/Med1 in a cell cycle-dependent manner, resulting in peak levels of expression during the G2/M phase of the cell cycle. ERK phosphorylation of ectopic TRAP220/Med1 also triggered shuttling into the nucleolus, thus suggesting that ERK may regulate TRAP220/Med1 subnuclear localization. Finally, we observed that ERK phosphorylation of TRAP220/Med1 stimulates its intrinsic transcriptional coactivation activity. We propose that ERK-mediated phosphorylation is a regulatory mechanism that controls TRAP220/Med1 expression levels and modulates its functional activity.


2003 ◽  
Vol 23 (6) ◽  
pp. 1896-1909 ◽  
Author(s):  
Ju-Ming Wang ◽  
Ming-Zong Lai ◽  
Hsin-Fang Yang-Yen

ABSTRACT We have previously demonstrated that the antiapoptotic gene mcl-1 is activated by interleukin-3 (IL-3) in Ba/F3 pro-B cells through two promoter elements designated the CRE-2 and SIE motifs. While the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through the phosphatidylinositol 3-kinase/Akt-dependent pathway, the identity and cytokine activation pathway of the SIE-binding complex remains unclear. In this report, we demonstrated that PU.1 is one component of the SIE-binding complex. A chromatin immunoprecipitation assay further confirmed that PU.1 binds to the mcl-1 promoter region containing the SIE motif in vivo. While IL-3 stimulation does not significantly alter the SIE-binding activity of PU.1, it markedly increases PU.1's transactivation activity. The latter effect coincides with the increased phosphorylation of PU.1 following IL-3 activation of a p38 mitogen-activated protein kinase (p38MAPK)-dependent pathway. A serine-to-alanine substitution at position 142 significantly weakens PU.1's ability to be phosphorylated by the p38MAPK immunocomplex. Furthermore, this S142A mutant is impaired in the ability to be further stimulated by IL-3 to transactivate the mcl-1 reporter through the SIE motif. Taken together, our results demonstrate that IL-3 stimulation of mcl-1 gene transcription through the SIE motif involves phosphorylation of PU.1 at serine 142 by a p38MAPK-dependent pathway.


2013 ◽  
Vol 111 (3) ◽  
pp. 452-464 ◽  
Author(s):  
Gina Cecilia Pistol ◽  
Mihail Alexandru Gras ◽  
Daniela Eliza Marin ◽  
Florentina Israel-Roming ◽  
Mariana Stancu ◽  
...  

Zearalenone (ZEA) is an oestrogenic mycotoxin produced byFusariumspecies, considered to be a risk factor from both public health and agricultural perspectives. In the presentin vivostudy, a feeding trial was conducted to evaluate thein vivoeffect of a ZEA-contaminated diet on immune response in young pigs. The effect of ZEA on pro-inflammatory (TNF-α, IL-8, IL-6, IL-1β and interferon-γ) and anti-inflammatory (IL-10 and IL-4) cytokines and other molecules involved in inflammatory processes (matrix metalloproteinases (MMP)/tissue inhibitors of matrix metalloproteinases (TIMP), nuclear receptors: PPARγ and NF-κB1, mitogen-activated protein kinases (MAPK): mitogen-activated protein kinase kinase kinase 7 (TAK1)/mitogen-activated protein kinase 14 (p38α)/mitogen-activated protein kinase 8 (JNK1)/ mitogen-activated protein kinase 9 (JNK2)) in the liver of piglets was investigated. The present results showed that a concentration of 316 parts per billion ZEA leads to a significant decrease in the levels of pro- and anti-inflammatory cytokines at both gene expression and protein levels, correlated with a decrease in the levels of other inflammatory mediators, MMP and TIMP. The results also showed that dietary ZEA induces a dramatic reduction in the expressions ofNF-κB1andTAK1/p38αMAPK genes in the liver of the experimentally intoxicated piglets, and has no effect on the expression ofPPARγmRNA. The present results suggest that the toxic action of ZEA begins in the upstream of the MAPK signalling pathway by the inhibition of TAK1, a MAPK/NF-κB activator. In conclusion, the present study shows that ZEA alters several important parameters of the hepatic cellular immune response. From an economic point of view, these data suggest that, in pigs, ZEA is not only a powerful oestrogenic mycotoxin but also a potential hepatotoxin when administered through the oral route. Therefore, the present results represent additional data from cellular and molecular levels that could be taken into account in the determination of the regulation limit of the tolerance to ZEA.


2009 ◽  
Vol 181 (4) ◽  
pp. 659
Author(s):  
Lakshmipathi Khandrika ◽  
Binod Kumar ◽  
Sweaty Koul ◽  
Randall B Meacham ◽  
Hari K Koul

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