scholarly journals Activation of TRAP/Mediator Subunit TRAP220/Med1 Is Regulated by Mitogen-Activated Protein Kinase-Dependent Phosphorylation

2005 ◽  
Vol 25 (24) ◽  
pp. 10695-10710 ◽  
Author(s):  
Pradeep K. Pandey ◽  
T. S. Udayakumar ◽  
Xinjie Lin ◽  
Dipali Sharma ◽  
Paul S. Shapiro ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Specific Substrate ◽  
Dependent Manner ◽  
Erk Phosphorylation ◽  
Mitogen Activated Protein

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.

scholarly journals MED1 Phosphorylation Promotes Its Association with Mediator: Implications for Nuclear Receptor Signaling

2008 ◽  
Vol 28 (12) ◽  
pp. 3932-3942 ◽  
Author(s):  
Madesh Belakavadi ◽  
Pradeep K. Pandey ◽  
Ravi Vijayvargia ◽  
Joseph D. Fondell
Keyword(s):  
Rna Polymerase Ii ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Nuclear Hormone Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Erk Phosphorylation ◽  
Broad Array ◽  
Mitogen Activated Protein

ABSTRACT Mediator is a conserved multisubunit complex that acts as a functional interface between regulatory transcription factors and the general RNA polymerase II initiation apparatus. MED1 is a pivotal component of the complex that binds to nuclear receptors and a broad array of other gene-specific activators. Paradoxically, MED1 is found in only a fraction of the total cellular Mediator complexes, and the mechanisms regulating its binding to the core complex remain unclear. Here, we report that phosphorylation of MED1 by mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) promotes its association with Mediator. We show that MED1 directly binds to the MED7 subunit and that ERK phosphorylation of MED1 enhances this interaction. Interestingly, we found that both thyroid and steroid hormones stimulate MED1 phosphorylation in vivo and that MED1 phosphorylation is required for its nuclear hormone receptor coactivator activity. Finally, we show that MED1 phosphorylation by ERK enhances thyroid hormone receptor-dependent transcription in vitro. Our findings suggest that ERK phosphorylation of MED1 is a regulatory mechanism that promotes MED1 association with Mediator and, as such, may facilitate a novel feed-forward action of nuclear hormones.

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 Activation of p42 Mitogen-activated Protein Kinase (MAPK), but not c-Jun NH2-Terminal Kinase, Induces Phosphorylation and Stabilization of MAPK Phosphatase XCL100 inXenopus Oocytes

2002 ◽  
Vol 13 (2) ◽  
pp. 454-468 ◽  
Author(s):  
Michael L. Sohaskey ◽  
James E. Ferrell
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Limited Information ◽  
Mapk Activity ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Mapk Phosphatase

Dual-specificity protein phosphatases are implicated in the direct down-regulation of mitogen-activated protein kinase (MAPK) activity in vivo. Accumulating evidence suggests that these phosphatases are components of negative feedback loops that restore MAPK activity to low levels after diverse physiological responses. Limited information exists, however, regarding their posttranscriptional regulation. We cloned two Xenopus homologs of the mammalian dual-specificity MAPK phosphatases MKP-1/CL100 and found that overexpression of XCL100 in G2-arrested oocytes delayed or prevented progesterone-induced meiotic maturation. Epitope-taggedXCL100 was phosphorylated on serine during G2 phase, and on serine and threonine in a p42 MAPK-dependent manner during M phase. Threonine phosphorylation mapped to a single residue, threonine 168. Phosphorylation of XCL100 had no measurable effect on its ability to dephosphorylate p42 MAPK. Similarly, mutation of threonine 168 to either valine or glutamate did not significantly alter the binding affinity of a catalytically inactive XCL100 protein for active p42 MAPK in vivo. XCL100 was a labile protein in G2-arrested and progesterone-stimulated oocytes; surprisingly, its degradation rate was increased more than twofold after exposure to hyperosmolar sorbitol. In sorbitol-treated oocytes expressing a conditionally active ΔRaf-DD:ER chimera, activation of the p42 MAPK cascade led to phosphorylation of XCL100 and a pronounced decrease in the rate of its degradation. Our results provide mechanistic insight into the regulation of a dual-specificity MAPK phosphatase during meiotic maturation and the adaptation to cellular stress.

Involvement of the Mitogen-activated Protein Kinase Family in Tetracaine-induced PC12 Cell Death

2002 ◽  
Vol 96 (5) ◽  
pp. 1191-1201 ◽  
Author(s):  
Zhiming Tan ◽  
Shuji Dohi ◽  
Jinen Chen ◽  
Yosiko Banno ◽  
Yoshinori Nozawa
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Dna Fragmentation ◽  
Local Anesthetics ◽  
Caspase 3 ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Erk Phosphorylation ◽  
Mitogen Activated Protein

Background To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis. Methods Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye. Results Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Caspase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis. Conclusions Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaine's action for apoptosis.

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 MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signalling

2005 ◽  
Vol 390 (3) ◽  
pp. 749-759 ◽  
Author(s):  
Joanne Darragh ◽  
Ana Soloaga ◽  
Victoria A. Beardmore ◽  
Andrew D. Wingate ◽  
Giselle R. Wiggin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Early Gene ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Activator Protein 1 ◽  
Double Knockout ◽  
Mitogen Activated Protein

MSK (mitogen- and stress-activated protein kinase) 1 and MSK2 are kinases activated downstream of either the ERK (extracellular-signal-regulated kinase) 1/2 or p38 MAPK (mitogen-activated protein kinase) pathways in vivo and are required for the phosphorylation of CREB (cAMP response element-binding protein) and histone H3. Here we show that the MSKs are involved in regulating the transcription of the immediate early gene Nur77. Stimulation of mouse embryonic fibroblasts with PMA, EGF (epidermal growth factor), TNF (tumour necrosis factor) or anisomycin resulted in induction of the Nur77 mRNA. The induction of Nur77 by TNF and anisomycin was abolished in MSK1/2 double-knockout cells, whereas induction was significantly reduced in response to PMA or EGF. The MSK responsive elements were mapped to two AP (activator protein)-1-like elements in the Nur77 promoter. The induction of Nur77 was also blocked by A-CREB, suggesting that MSKs control Nur77 transcription by phosphorylating CREB bound to the two AP-1-like elements. Consistent with the decrease in Nur77 mRNA levels in the MSK1/2-knockout cells, it was also found that MSKs were required for the induction of Nur77 protein by PMA and TNF. MSKs were also found to be required for the transcription of two genes related to Nur77, Nurr1 and Nor1, which were also transcribed in a CREB- or ATF1 (activating transcription factor-1)-dependent manner. Downstream of anisomycin signalling, a second ERK-dependent pathway, independent of MSK and CREB, was also required for the transcription of Nurr1 and Nor1.

scholarly journals Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

2005 ◽  
Vol 392 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Mercedes Pozuelo Rubio ◽  
David G. Campbell ◽  
Nicholas A. Morrice ◽  
Carol Mackintosh
Keyword(s):  
Protein Kinase ◽  
Metal Ion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

scholarly journals Rck2 Kinase Is a Substrate for the Osmotic Stress-Activated Mitogen-Activated Protein Kinase Hog1

2000 ◽  
Vol 20 (11) ◽  
pp. 3887-3895 ◽  
Author(s):  
Elizabeth Bilsland-Marchesan ◽  
Joaquín Ariño ◽  
Haruo Saito ◽  
Per Sunnerhagen ◽  
Francesc Posas
Keyword(s):  
Protein Kinase ◽  
Osmotic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Mapk Activation ◽  
Mitogen Activated Protein ◽  
Two Hybrid

ABSTRACT Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Δ and pbs2Δ cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Δ cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.

scholarly journals Mitogen-activated protein kinase mediates the apoptosis of highly metastatic human non-small cell lung cancer cells induced by isothiocyanates

2011 ◽  
Vol 106 (12) ◽  
pp. 1779-1791 ◽  
Author(s):  
Huiqin Yan ◽  
Yu Zhu ◽  
Boning Liu ◽  
Heng Wu ◽  
Yongwen Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Cancer Cell ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Cell Metastasis ◽  
Mitogen Activated Protein ◽  
Dose Dependent

Dietary isothiocyanates have been shown to possess anti-tumour activity, inhibiting several types of cultured human cancer cell growth. However, there are limited studies on their effects on cancer cell metastasis. Our previous study showed that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) suppressed human lung cancer cell metastasis potential. In the present study, we found BITC (7·5 and 10 μm) and PEITC (12·5 and 20 μm) induced highly metastatic human non-small cell lung cancer L9981 cell apoptosis in a dose-dependent manner. Caspase-3 was activated. They also caused cell cycle arrest at the G2/M phase, via modulation of cyclin B1 expression. The mitogen-activated protein kinase (MAPK) signalling pathway was involved. c-Jun N-terminal kinase, extracellular signal-regulated protein kinase 1/2 and p38 were activated in a dose-dependent manner; activator protein 1 (AP-1) transcriptional activation and cyclin D1 expression were repressed. Apoptosis and MAPK activation were abrogated by anti-oxidant N-acetyl cysteine (NAC), suggesting that cell death signalling was triggered by oxidative stress. Further microarray analysis evaluated the potential targeted genes related to apoptosis and the cell cycle. Our studies suggested that BITC and PEITC suppressed the metastasis potential of highly metastatic lung cancer cells by inducing apoptosis and cell cycle arrest, via targeting the MAPK/AP-1 pathway. This may provide a novel approach for metastasis therapy of lung cancer by dietary isothiocyanates and possibly other types of cancer.

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