scholarly journals Monitoring protein–protein interactions in mammalian cells by trans-SUMOylation

2011 ◽  
Vol 438 (3) ◽  
pp. 495-503 ◽  
Author(s):  
Ratnesh K. Srivastav ◽  
Susan Schwede ◽  
Malte Klaus ◽  
Jessica Schwermann ◽  
Matthias Gaestel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Glutathione Transferase ◽  
Mitogen Activated Protein Kinase ◽  
Protein Protein Interactions ◽  
Sumoylation Site ◽  
Endogenous Expression ◽  
Mitogen Activated Protein

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.

Receptor mechanisms of rapid extranuclear signalling initiated by steroid hormones

2004 ◽  
Vol 40 ◽  
pp. 105-120 ◽  
Author(s):  
Viroj Boonyaratanakornkit ◽  
Dean P Edwards
Keyword(s):  
Protein Kinase ◽  
Steroid Hormones ◽  
Gene Transcription ◽  
Mammalian Cells ◽  
Hormone Receptors ◽  
Direct Interaction ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Protein Kinase Signalling

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.

scholarly journals Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2

2004 ◽  
Vol 380 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Guadalupe SABIO ◽  
Suzana REUVER ◽  
Carmen FEIJOO ◽  
Masato HASEGAWA ◽  
Gareth M. THOMAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Protein Interactions ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Cellular Stresses ◽  
Sb 203580

SAPK3 (stress-activated protein kinase-3, also known as p38γ) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of α1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38γ and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38γ binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38γ to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β but not SAPK3/p38γ, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38γ with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38γ co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38γ, a first physiological substrate described for SAPK3/p38γ and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein–protein interactions at the synapse in response to adverse stress- or mitogenrelated stimuli.

scholarly journals Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression

1999 ◽  
Vol 19 (1) ◽  
pp. 229-240 ◽  
Author(s):  
Angela M. Cacace ◽  
Neil R. Michaud ◽  
Marc Therrien ◽  
Karen Mathes ◽  
Terry Copeland ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Ras Signaling ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Mitogen Activated Protein

ABSTRACT Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. InXenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

scholarly journals Mutational Analysis of STE5 in the Yeast Saccharomyces cerevisiae: Application of a Differential Interaction Trap Assay for Examining Protein-Protein Interactions

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 479-492 ◽  
Author(s):  
Carla Inouye ◽  
Namrita Dhillon ◽  
Tim Durfee ◽  
Patricia C Zambryski ◽  
Jeremy Thorner
Keyword(s):  
Protein Interactions ◽  
Mutational Analysis ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitogen Activated Protein

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein, β subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5Δ cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

scholarly journals Natural feed contaminant zearalenone decreases the expressions of important pro- and anti-inflammatory mediators and mitogen-activated protein kinase/NF-κB signalling molecules in pigs

2013 ◽  
Vol 111 (3) ◽  
pp. 452-464 ◽  
Author(s):  
Gina Cecilia Pistol ◽  
Mihail Alexandru Gras ◽  
Daniela Eliza Marin ◽  
Florentina Israel-Roming ◽  
Mariana Stancu ◽  
...  
Keyword(s):  
Immune Response ◽  
Protein Kinase ◽  
Matrix Metalloproteinases ◽  
Inflammatory Mediators ◽  
Interferon Γ ◽  
Mitogen Activated Protein Kinase ◽  
Economic Point ◽  
Mitogen Activated Protein ◽  
Anti Inflammatory

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.

IN VIVO ACTIVATION OF P38 MITOGEN ACTIVATED PROTEIN KINASE SIGNALING CASCADE IN A HYPEROXALURIC RAT MODEL

2009 ◽  
Vol 181 (4) ◽  
pp. 659
Author(s):  
Lakshmipathi Khandrika ◽  
Binod Kumar ◽  
Sweaty Koul ◽  
Randall B Meacham ◽  
Hari K Koul
Keyword(s):  
Protein Kinase ◽  
Rat Model ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascade ◽  
Kinase Signaling ◽  
Mitogen Activated Protein ◽  
Protein Kinase Signaling
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