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 The Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases MK2 and MK3 Cooperate in Stimulation of Tumor Necrosis Factor Biosynthesis and Stabilization of p38 MAPK

2006 ◽  
Vol 27 (1) ◽  
pp. 170-181 ◽  
Author(s):  
N. Ronkina ◽  
A. Kotlyarov ◽  
O. Dittrich-Breiholz ◽  
M. Kracht ◽  
E. Hitti ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tumor Necrosis Factor ◽  
Protein Kinases ◽  
Tumor Necrosis ◽  
Mitogen Activated Protein Kinase ◽  
Double Knockout ◽  
Mitogen Activated Protein ◽  
Deficient Mice ◽  
Necrosis Factor

ABSTRACT MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.

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.

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 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 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 Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion

2019 ◽  
Vol 294 (46) ◽  
pp. 17395-17408 ◽  
Author(s):  
Nicola M. Blythe ◽  
Katsuhiko Muraki ◽  
Melanie J. Ludlow ◽  
Vasili Stylianidis ◽  
Hamish T. J. Gilbert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Cardiac Fibroblasts ◽  
Ruthenium Red ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mitogen Activated Protein

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/− cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.

PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli

2013 ◽  
Vol 452 (3) ◽  
pp. 531-543 ◽  
Author(s):  
Laura Novellasdemunt ◽  
Laurent Bultot ◽  
Anna Manzano ◽  
Francesc Ventura ◽  
Jose Luis Rosa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cancer Cells ◽  
Serum Response Factor ◽  
Gene Promoter ◽  
Mitogen Activated Protein Kinase ◽  
Early Gene ◽  
Mrna Levels ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Serum Response

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H2O2, UV radiation and anisomycin) leads to a rapid increase (15–30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (−918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells.

scholarly journals Estrogen Activation of Cyclic Adenosine 5′-Monophosphate Response Element-Mediated Transcription Requires the Extracellularly Regulated Kinase/Mitogen-Activated Protein Kinase Pathway

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 832-838 ◽  
Author(s):  
Christian B. Wade ◽  
Daniel M. Dorsa
Keyword(s):  
Protein Kinase ◽  
Mapk Pathway ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Mitogen Activated Protein ◽  
Element Binding Protein

The ability of estrogen to rapidly initiate a variety of signal transduction cascades is increasingly recognized as playing an important role in a number of tissue-specific transcriptional actions of the hormone. In vivo, estrogen rapidly elicits phosphorylation of cAMP response element-binding protein (CREB). We have previously shown that both ERα and ERβ are capable of activating the MAPK pathway in response to a low dose of 17β-estradiol. In the present study, the ability of estrogen to act through both ERα and ERβ to increase CREB phosphorylation was evaluated in an immortalized hippocampal cell line stably expressing either receptor. Estrogen treatment promoted rapid CREB phosphorylation, reaching a maximum by 15 min. This activation is completely blocked by the antiestrogen ICI 182,780, suggesting an estrogen receptor-dependent mechanism. The addition of the mitogen/ERK kinase-1 inhibitor, PD98059, also blocked the ability of estrogen to signal to CREB phosphorylation. Estrogen also caused an increase in p90Rsk activity, a critical mediator of MAPK effects. Surprisingly, blockade of the protein kinase A pathway in cells treated with estrogen did not affect estrogen-mediated CREB phosphorylation. Thus, MAPK and p90Rsk appear to be the primary mediators of estrogen-induced gene transcription through ERα and ERβ.

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