The interaction of p38 with its upstream kinase MKK6

Phosphoinositide-dependent protein kinase 1 (PDK1) is known to regulate PAR4 induced platelet activation and thrombus formation through GSK3β. However, whether PDK1 signaling also involves the ADP receptor and, if so, downstream functional consequences are unknown. We employed both pharmacologic (e.g. the selective PDK1 inhibitor, BX795) and genetic (platelet specific deletion of PDK1) approaches to dissect the role of PDK1 in ADP-induced platelet activation and protein synthesis. Inhibition of PDK1 with BX795 reduced 2MeSADP-induced platelet aggregation by abolishing thromboxane generation. Similar results were observed in PDK1 -/- mice (Fig A). Inhibition of PDK1 protected mice from collagen and epinephrine-induced pulmonary embolism (Fig B). PDK1 was also necessary for the phosphorylation of MEK1/2, Erk1/2 and cPLA2, indicating that PDK1 regulates an upstream kinase in MAPK pathway. We next identified that this upstream kinase is Raf1 (necessary for the phosphorylation of MEK1/2), as pharmacologic inhibition and genetic ablation of PDK1 was sufficient to prevent Raf1 phosphorylation (Fig C). Pharmacologic inhibition and genetic ablation of PDK1 blocked MAPK- and mTORC1-dependent protein synthesis in platelets through a mechanism requiring the phosphorylation of eIF4E and S6K. Concordantly, PDK1 is necessary for signal-dependent synthesis of the protein bcl3, which is under mTORC1-dependent control (Fig C). Taken together, our findings show for the first time that PDK1, a master kinase in the PI3K pathway, directly governs thromboxane generation, thrombosis, and protein synthesis in platelets through regulating MAPK and mTORC1 pathways.

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Abstract 186: Hdac7-derived 7aa Peptide May Function as a Phosphorylation Carrier

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.186 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Junyao Yang ◽

Wen Wang ◽

Qian Wang ◽

Lingfang Zeng

Keyword(s):

Endothelial Cell ◽

Vascular Endothelial Cell ◽

Blood Perfusion ◽

Open Reading Frames ◽

In Vivo Studies ◽

Buffer System ◽

Vascular Progenitor Cells ◽

Upstream Kinase

Background: Histone deacetylase 7 (HDAC7) belongs to class II HDAC family, playing a pivotal role in the maintenance of endothelium integrity. There are 8 splicing variants in mouse HDAC7 mRNAs. Within the 5’ terminal non-coding area of some variants, there exist some short open reading frames (sORFs). Whether these sORFs can be translated and their potential roles in cellular physiology remain unclear. Method and results: Our previous studies suggested that one mouse HDAC7 produced a 7aa peptide from the non-coding area. In this study, we demonstrated that one sORF encoding a 7 amino acids (aa)-peptide could be translated in response to vascular endothelial cell growth factor (VEGF) in vascular progenitor cells (VPCs). The 7aa-peptide (7A) could be phosphorylated at serine residue via MEKK1. Importantly, the phosphorylated 7aa-peptide (7Ap) could transfer the phosphorylation group to the Thr residue of the 14-3-3γ protein in a cell free in-gel buffer system. The in vitro functional analyses revealed that 7A enhanced VEGF-induced VPC migration and differentiation toward endothelial cell (EC) lineage, in which MEKK1 and 14-3-3γ served as upstream kinase and downstream effector respectively. Knockdown of either MEKK1 or 14-3-3γ attenuated VEGF-induced VPC migration and differentiation. Exogenous 7Ap could rescue VEGF effect in MEKK1 but not in 14-3-3γ knockdown cells. The in vivo studies showed that 7A especially 7Ap induced capillary vessel formation within matrigel plug assays, increased re-endothelialization and suppressed neointima formation in the femoral artery injury model, and promoted the foot blood perfusion recovery in the hindlimb ischemia model. Conclusion: These results indicate that the sORFs within the non-coding area can be translated under some circumstances and that the 7aa-peptide may play an important role in cellular processes like migration and differentiation via acting as a phosphorylation carrier. Significance: As a phosphorylation carrier, 7aa possesses therapeutic potentials in tackling angiogenesis related diseases.

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Abstract 438: Hydrogen Sulfide Promotes Mitochondrial Biogenesis Through the Regulation of AMP-activated Protein Kinase

Circulation Research ◽

10.1161/res.119.suppl_1.438 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Yuuki Shimizu ◽

Rohini Polavarapu ◽

John Calvert

Keyword(s):

Hydrogen Sulfide ◽

Mitochondrial Biogenesis ◽

Target Genes ◽

Nuclear Dna ◽

Citrate Synthase ◽

Serine Phosphorylation ◽

Signaling Cascade ◽

Disease States ◽

Upstream Kinase ◽

High Concentrations

Background: Hydrogen sulfide (H 2 S) possesses numerous cellular actions that account for its cardioprotective effects. A mechanism of particular interest is its effects on the mitochondria. At low concentrations, H 2 S donates electrons to the electron transport chain, whereas at high concentrations it inhibits mitochondrial respiration. H 2 S therapy improves mitochondrial function and prevents the loss of mitochondria following the onset of myocardial ischemia. However, it is not known if these improvements are associated with simply a reduction in injury or if mitochondrial biogenesis is involved. Therefore, the purpose of this study was to determine if H 2 S regulates/induces mitochondrial biogenesis in the heart. Methods and Results: C57BL/6J mice (8 weeks of age) were given an orally active H 2 S donor (SG-1002; 20 mg/kg/day) in their chow for 4 weeks. For these studies we focused our analysis on an AMPK-PGC1α signaling cascade. SG-1002 significantly increased the phosphorylation of AMPK, the serine phosphorylation of PGC1α, and increased the nuclear localization of PGC1α. This was associated with an increase in the gene expression of PGC1α target genes associated with mitochondrial biogenesis, an increase in mitochondrial to nuclear DNA ratios and an increase in citrate synthase activity. SG-1002 failed to elicit these changes in AMPK deficient mice. Therefore, we sought to determine how SG-1002 activated AMPK. SG-1002 did not alter the phosphorylation of LKB1, an upstream kinase of AMPK, and did not alter the levels of AMP (activator of AMPK). SG-1002 did not alter the expression of protein phosphatase 2A (PP2A; dephosphorylates AMPK), but it did significantly decrease the activity of PP2A). This decrease was accompanied by an increase in the sulfhydration of PP2A, suggesting that this modification is inhibitory. Conclusion: These data suggest that H 2 S augments mitochondrial biogenesis in the heart via an AMPK-PGC1α signaling cascade. This is important because mitochondrial abnormalities are associated with a number of disease states (diabetes and heart failure) where H 2 S levels are decreased. Therefore, strategies aimed at increasing H 2 S levels could potentially induce the generation of new, healthy mitochondria.

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TYK2: An Upstream Kinase of STATs in Cancer

Cancers ◽

10.3390/cancers11111728 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1728 ◽

Cited By ~ 5

Author(s):

Katharina Wöss ◽

Natalija Simonović ◽

Birgit Strobl ◽

Sabine Macho-Maschler ◽

Mathias Müller

Keyword(s):

Protein Tyrosine Kinase ◽

Inflammatory Responses ◽

Immune Surveillance ◽

Pharmacological Intervention ◽

Cytokine Signaling ◽

Loss Of Function ◽

Signal Transducers ◽

Protein Levels ◽

Tyrosine Kinase 2 ◽

Upstream Kinase

In this review we concentrate on the recent findings describing the oncogenic potential of the protein tyrosine kinase 2 (TYK2). The overview on the current understanding of TYK2 functions in cytokine responses and carcinogenesis focusses on the activation of the signal transducers and activators of transcription (STAT) 3 and 5. Insight gained from loss-of-function (LOF) gene-modified mice and human patients homozygous for Tyk2/TYK2-mutated alleles established the central role in immunological and inflammatory responses. For the description of physiological TYK2 structure/function relationships in cytokine signaling and of overarching molecular and pathologic properties in carcinogenesis, we mainly refer to the most recent reviews. Dysregulated TYK2 activation, aberrant TYK2 protein levels, and gain-of-function (GOF) TYK2 mutations are found in various cancers. We discuss the molecular consequences thereof and briefly describe the molecular means to counteract TYK2 activity under (patho-)physiological conditions by cellular effectors and by pharmacological intervention. For the role of TYK2 in tumor immune-surveillance we refer to the recent Special Issue of Cancers “JAK-STAT Signaling Pathway in Cancer”.

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Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00558.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. H2412-H2421 ◽

Cited By ~ 25

Author(s):

Markus Frederich ◽

Li Zhang ◽

James A. Balschi

Keyword(s):

Protein Kinase ◽

Metabolic Inhibitors ◽

Protein Kinase Activity ◽

Α Subunit ◽

Rat Hearts ◽

Upstream Kinase ◽

Ampk Activity ◽

Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

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AMPK α2 subunit is involved in platelet signaling, clot retraction, and thrombus stability

Blood ◽

10.1182/blood-2010-04-279612 ◽

2010 ◽

Vol 116 (12) ◽

pp. 2134-2140 ◽

Cited By ~ 30

Author(s):

Voahanginirina Randriamboavonjy ◽

Johann Isaak ◽

Timo Frömel ◽

Benoit Viollet ◽

Beate Fisslthaler ◽

...

Keyword(s):

Kinase Inhibitors ◽

Adenosine Monophosphate ◽

Human Platelets ◽

Whole Body ◽

Initial Increase ◽

Clot Retraction ◽

Compound C ◽

Platelet Signaling ◽

Upstream Kinase

Abstract The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca2+]i. Clot retraction was also impaired in platelets from AMPKα2−/− mice but not from wild-type littermates or AMPKα1−/− mice. Moreover, rebleeding was more frequent in AMPKα2−/− mice, and the FeCl3-induced thrombi formed in AMPKα2−/− mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2—by affecting Fyn phosphorylation and activity—plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.

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The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

Eukaryotic Cell ◽

10.1128/ec.00018-15 ◽

2015 ◽

Vol 14 (6) ◽

pp. 602-615 ◽

Cited By ~ 13

Author(s):

Bert-Ewald Priegnitz ◽

Ulrike Brandt ◽

Khomaizon A. K. Pahirulzaman ◽

Jeroen S. Dickschat ◽

André Fleißner

Keyword(s):

Aspergillus Niger ◽

Map Kinase ◽

Secondary Metabolism ◽

Filamentous Fungi ◽

Cell Types ◽

Growth Conditions ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

Mitogen Activated Protein ◽

Upstream Kinase

ABSTRACTAdaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black moldAspergillus niger(AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation inA. nigeris triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

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AMPK: A Key Sensor of Fuel and Energy Status in Skeletal Muscle

Physiology ◽

10.1152/physiol.00044.2005 ◽

2006 ◽

Vol 21 (1) ◽

pp. 48-60 ◽

Cited By ~ 294

Author(s):

D. Grahame Hardie ◽

Kei Sakamoto

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Signaling Pathway ◽

Recent Work ◽

Metabolic Changes ◽

Energy Status ◽

Upstream Kinase ◽

Amp Activated Protein Kinase

Contraction induces marked metabolic changes in muscle, and the AMP-activated protein kinase (AMPK) is a good candidate to explain these effects. Recent work using a muscle-specific knockout of the upstream kinase, LKB1, has confirmed that the LKB1→AMPK cascade is the signaling pathway responsible for many of these effects.

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Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells

Journal of Endocrinology ◽

10.1677/joe.0.1720271 ◽

2002 ◽

Vol 172 (2) ◽

pp. 271-281 ◽

Cited By ~ 19

Author(s):

H Tokuda ◽

O Kozawa ◽

M Niwa ◽

H Matsuno ◽

K Kato ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Prostaglandin E2 ◽

Map Kinase ◽

Specific Inhibitor ◽

Heat Shock Protein 27 ◽

P38 Map Kinase ◽

Calphostin C ◽

Mitogen Activated Protein ◽

Upstream Kinase

We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

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