Supranutritional selenium induces alterations in molecular targets related to energy metabolism in skeletal muscle and visceral adipose tissue of pigs

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern that supranutritional Se intake may increase the risk to develop Type 2 diabetes mellitus (T2DM). We aimed to determine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivity and signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg; high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantly increased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. In skeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead box O1 transcription factor and peroxisomal proliferator-activated receptor- coactivator 1 were increased and gene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Se pigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphorylation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism in major insulin target tissues, though this is probably not sufficient to induce diabetes.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

Collagen or collagen-related peptide cause (Ca2+)i elevation and increased tyrosine phosphorylation in human megakaryocytes.

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.

We have recently shown that collagen activates platelets through a pathway dependent on the Fc receptor gamma-chain and the tyrosine kinase Syk. We report here that the Fc receptor gamma-chain and the candidate collagen receptor glycoprotein VI (GPVI) co-associate. Furthermore, cross-linking GPVI stimulates a similar pattern of tyrosine phosphorylation to that stimulated by collagen, including tyrosine phosphorylation of Fc receptor gamma-chain. These results support a model where GPVI couples collagen-stimulation of platelets to phosphorylation of the Fc receptor gamma-chain leading to activation of Syk and phospholipase Cgamma2.

A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1.

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor gamma chain, the tyrosine kinase syk, and phospholipase C gamma2 (PLC gamma2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support alpha2beta1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin alpha2beta1. This finding suggests the existence of a platelet receptor other than alpha2beta1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLC gamma2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin alpha2beta1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLC gamma2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of alpha2beta1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an alpha2beta1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLC gamma2. This pathway appears to contribute to platelet activation by collagen.

Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis

Glycogen synthase kinase 3 (GSK3, of which there are two isoforms, GSK3alpha and GSK3beta) was originally characterized in the context of regulation of glycogen metabolism, though it is now known to regulate many other cellular processes. Phosphorylation of GSK3alpha(Ser21) and GSK3beta(Ser9) inhibits their activity. In the heart, emphasis has been placed particularly on GSK3beta, rather than GSK3alpha. Importantly, catalytically-active GSK3 generally restrains gene expression and, in the heart, catalytically-active GSK3 has been implicated in anti-hypertrophic signalling. Inhibition of GSK3 results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses, and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3 passes primarily through protein kinase B/Akt (PKB/Akt). However, recent data suggest that the situation is far more complex. We review evidence pertaining to the role of GSK3 in the myocardium and discuss effects of genetic manipulation of GSK3 activity in vivo. We also discuss the signalling pathways potentially regulating GSK3 activity and propose that, depending on the stimulus, phosphorylation of GSK3 is independent of PKB/Akt. Potential GSK3 substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells, beta-catenin, GATA4, myocardin, CREB, and eukaryotic initiation factor 2Bvarepsilon. These and other transcription factor substrates putatively important in the heart are considered. We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3 level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3 in cardiac processes.

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Antithrombotic actions of statins involve PECAM-1 signalling

Statins are widely prescribed cholesterol-lowering drugs that are a first-line treatment for coronary artery disease and atherosclerosis, reducing the incidence of thrombotic events such as myocardial infarction and stroke. Statins have been shown to reduce platelet activation, although the mechanism(s) through which this occurs is unclear. Since several of the characteristic effects of statins on platelets are shared with those elicited by the inhibitory platelet adhesion receptor PECAM-1, we investigated a potential connection between the influence of statins on platelet function and PECAM-1 signalling. Statins were found to inhibit a range of platelet functional responses and thrombus formation in vitro and in vivo. Notably, these effects of statins on platelet function in vitro and in vivo were diminished in PECAM-1-/- platelets. Activation of PECAM-1 signalling results in its tyrosine phosphorylation, the recruitment and activation of tyrosine phosphatase SHP-2, the subsequent binding of phosphoinositol 3-kinase (PI3-K) and diminished PI3-K signalling. Statins resulted in the stimulation of these events, leading to the inhibition of Akt activation. Together, these data provides evidence for a fundamental role of PECAM-1 in the inhibitory effects of statins on platelet activation, which may explain some of the pleiotropic actions of these drugs.

Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine

Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered.

Pharmacological actions of nobiletin in the modulation of platelet function

Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. The fibrinogen binding, α-granule secretion and calcium mobilisation assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore nobiletin may represent a potential antithrombotic agent of dietary origins.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.