Vagal nerve stimulation improves mitochondrial dynamics via an M3 receptor/CaMKKβ/AMPK pathway in isoproterenol‐induced myocardial ischaemia

Mitochondrial dynamics—fission and fusion—are associated with ischaemic heart disease (IHD). This study explored the protective effect of vagal nerve stimulation (VNS) against isoproterenol (ISO)‐induced myocardial ischaemia in a rat model and tested whether VNS plays a role in preventing disorders of mitochondrial dynamics and function. Isoproterenol not only caused cardiac injury but also increased the expression of mitochondrial fission proteins [dynamin‐related peptide1 (Drp1) and mitochondrial fission protein1 (Fis‐1)) and decreased the expression of fusion proteins (optic atrophy‐1 (OPA1) and mitofusins1/2 (Mfn1/2)], thereby disrupting mitochondrial dynamics and leading to increase in mitochondrial fragments. Interestingly, VNS restored mitochondrial dynamics through regulation of Drp1, Fis‐1, OPA1 and Mfn1/2; enhanced ATP content and mitochondrial membrane potential; reduced mitochondrial permeability transition pore (MPTP) opening; and improved mitochondrial ultrastructure and size. Furthermore, VNS reduced the size of the myocardial infarction and ameliorated cardiomyocyte apoptosis and cardiac dysfunction induced by ISO. Moreover, VNS activated AMP‐activated protein kinase (AMPK), which was accompanied by phosphorylation of Ca²⁺/calmodulin‐dependent protein kinase kinase β (CaMKKβ) during myocardial ischaemia. Treatment with subtype‐3 of muscarinic acetylcholine receptor (M₃R) antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide or AMPK inhibitor Compound C abolished the protective effects of VNS on mitochondrial dynamics and function, suggesting that M₃R/CaMKKβ/AMPK signalling are involved in mediating beneficial effects of VNS. This study demonstrates that VNS modulates mitochondrial dynamics and improves mitochondrial function, possibly through the M₃R/CaMKKβ/AMPK pathway, to attenuate ISO‐induced cardiac damage in rats. Targeting mitochondrial dynamics may provide a novel therapeutic strategy in IHD.     

Nrf2 attenuates inflammatory response in COPD/emphysema: Crosstalk with Wnt3a/β‐catenin and AMPK pathways

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2^?/? mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.     

AMPKα2 knockout enhances tumour inflammation through exacerbated liver injury and energy deprivation‐associated AMPKα1 activation

Tissue damage and its associated‐inflammation act as tumour initiators or propagators. AMP‐activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour‐bearing liver and the enhanced‐hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour‐associated phenotype. Thus, the enhanced tumour‐associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background

Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. α_IIbβ₃ is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods

Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results

Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions

We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

     

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.     

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.     

Cordycepin activates AMP‐activated protein kinase (AMPK) via interaction with the γ1 subunit

Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP‐activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin‐induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)‐elicited intracellular lipid accumulation and increased AMPK activity in a dose‐dependent manner. Cordycepin‐induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin‐dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.     

Infusion of a Lipid Emulsion Modulates AMPK and Related Proteins in Rat Liver,Muscle, and Adipose Tissues

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate‐buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca²⁺/calmodulin‐dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCβ, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARγ‐co‐activator 1α (PGC1α) protein levels were also increased in LE‐treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCα, PPARα, AdipoR1, AdipoR2, and sterol regulatory element–binding protein‐1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.     

Berberine suppresses neuroinflammatory responses through AMP‐activated protein kinase activation in BV‐2 microglia

The AMPK cascade is a sensor of cellular energy change, which monitors the AMP/ATP ratio to regulate cellular metabolism by restoring ATP levels, but its regulation of neuroinflammation mechanism remains unclear. Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been shown to improve several metabolic disorders, such as obesity and type II diabetes. However, the effect of berberine on neuroinflammatory responses in microglia are poorly understood. This study shows that berberine represses proinflammatory responses through AMP‐activated protein kinase (AMPK) activation in BV‐2 microglia. Our findings also demonstrate that berberine significantly down‐regulates LPS‐ or interferon (IFN)‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) expression in BV‐2 microglia cells. Berberine also inhibited LPS‐ or IFN‐γ‐induced nitric oxide production. In addition, berberine effectively inhibited proinflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 expression. On the other hand, upon various inflammatory stimulus including LPS and IFN‐γ, berberine suppressed the phosphorylated of ERK but not p38 and JNK in BV‐2 microglia. AMPK activation is catalyzed by upstream kinases such as LKB1 and Ca2+/calmodulin‐dependent protein kinase kinase‐II (CaMKK II). Moreover, berberine induced LKB1 (Ser428), CaMKII (Thr286), and AMPK (Thr172) phosphorylation, but not AMPK (Ser485). Furthermore, the inhibitory effect of berberine on iNOS and COX‐2 expression was abolished by AMPK inhibition via Compound C, an AMPK inhibitor. Berberine‐suppressed ERK phosphorylation was also reversed by Compound C treatment. Our data demonstrate that berberine significantly induces AMPK signaling pathways activation, which is involved in anti‐neuroinflammation. J. Cell. Biochem. 110: 697–705, 2010. © 2010 Wiley‐Liss, Inc.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Phosphorylation of VASP by AMPK alters actin binding and occurs at a novel site

Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that functions in adhesion and migration. In epithelial cells, VASP participates in cell–cell adhesion. At the molecular level, VASP drives actin bundling and polymerization. VASP activity is primarily regulated by phosphorylation. Three physiologically relevant phosphorylation sites significantly reduce actin regulatory activity and are targeted by several kinases, most notable Abl and protein kinases A and G (PKA and PKG). AMP-dependent kinase (AMPK) is best characterized as a cellular sensor of ATP depletion, but also alters actin dynamics in epithelial cells and participates in cell polarity pathways downstream of LKB1. While little is known about how AMPK direct changes in actin dynamics, AMPK has been shown to phosphorylate VASP at one of these three well-characterized PKA/PKG phosphorylation sites. Here we show that phosphorylation of VASP by AMPK occurs at a novel site, serine 322, and that phosphorylation at this site alters actin filament binding. We also show that inhibition of AMPK activity results in the accumulation of VASP at cell–cell adhesions and a concomitant increase in cell–cell adhesion.     

Ca<Superscript>2+</Superscript>-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca²⁺ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca²⁺ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca²⁺ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca²⁺, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca²⁺ modulation in rat submandibular gland acinar cells. Ca²⁺-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.     

Activation of the AMP-activated protein kinase (AMPK) by nitrated lipids in endothelial cells

The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO(2)) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO(2) induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO(2)-induced AMPK phosphorylation. OA-NO(2) induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO(2)-induced HIF-1α upregulation, suggesting that OA-NO(2)/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO(2) enhanced endothelial intracellular Ca(2+), an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO(2)-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO(2) activates AMPK in endothelial cells via an HO-1-dependent mechanism that increases HIF-1α protein expression and Ca(2+)/CaMKKβ activation.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Proteomic identification of pleckstrin‐associated proteins in platelets: Possible interactions with actin

Pleckstrin (plek)‐null platelets from a knockout mouse have been shown to be defective in granule secretion, aggregation and actin polymerization. However, the mechanism of plek signaling is currently unknown. Therefore, we sought to identify plek‐binding proteins in platelets by using GST pulldown assays and immunoprecipitation to isolate proteins from extracts of protein kinase C‐activated or inhibited human platelets. Co‐purified plek‐binding proteins were resolved by SDS‐PAGE and identified via nanospray quadruple TOF MS. Identified proteins may be involved in various cellular processes including cytoskeletal reorganization (moesin, radixin and α‐actinin) and signal transduction (serum deprivation response protein, 17 β‐hydroxysteroid dehydrogenase 4 and factor XIIIA). Both platelet aggregation and/or secretion require actin polymerization. However, studies have shown no direct association between plek and actin. Based on our findings we propose indirect associations between plek and actin through 17 β‐hydroxysteroid dehydrogenase 4, α‐actinin, moesin, radixin and factor XIIIA, which in turn suggest new roles for plek in platelet biology.     

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

BackgroundCa²⁺/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca²⁺-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.MethodsAnti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.ResultsOur data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca²⁺/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme.ConclusioncAMP/PKA signaling may confer Ca²⁺-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.General significanceOur results suggest a novel cross-talk between cAMP/PKA and Ca²⁺/CaM/CaMKKβ signaling through regulatory phosphorylation.     

Signalling mechanisms of the leukocyte integrin αMβ2: Current and future perspectives

Integrins are a family of heterodimeric cell adhesion receptors expressed on most cells and are involved in many cellular functions including phagocytosis, a process by which professional phagocytes recognise, bind and internalise foreign materials larger than 0.5 µm in diameter. An example of a phagocytic integrin receptor is αMβ2, and this review seeks to provide fresh insights into the current knowledge of this subject. Key areas that this review will emphasise include, the classical understanding of bi‐directional signalling to and from αMβ2 (aka inside‐out and outside‐in signalling, respectively). For inside‐out signalling, we will review the involvement of the small GTPase, Rap1, FERM‐containing proteins such as talin and kindlin‐3, some of the kinases, and the GEF, cytohesin‐1 and vasodilator‐stimulated phosphoprotein (VASP). We also summarise studies into outside‐in signalling, focussing on the roles of RhoA and RhoG, and activation of Rac1 through the complex comprising TIAM, 14‐3‐3 and β2. We will also consider non‐classical signalling processes, which include integrin clustering and membrane ruffling. Through this review, we hope to highlight the importance of αMβ2 signalling mechanisms and their relevance to other integrin‐mediated events.     

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

The calcium-calmodulin–dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca²⁺, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser⁴⁹⁵ directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser¹⁰⁰ and Ser⁵¹¹ mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser⁴⁹⁵. We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser⁵¹¹) and noncanonical (Ser¹⁰⁰) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.