Elk-1 is a novel protein-binding partner for FAK, regulating phagocytosis in medfly hemocytes

Focal adhesion kinase (FAK) and its downstream signaling targets, mitogen-activated protein kinase (MAPKs), are implicated in the process of phagocytosis by insect hemocytes. The goal of this study was to explore further the signaling pathways underlining the process of phagocytosis. The combination of bioinformatics, biochemical, and immunofluorescence approaches strongly support the expression of Elk-1-like protein in medfly hemocytes. Elk-1 is phosphorylated in E. coli or latex beads-challenged hemocytes and osmotic loading experiments as well as flow cytometry analysis demonstrated that Elk-1-like protein regulates the uptake of bacteria. RNA interference (RNAi) and pharmacological inhibitors show that the signaling for Elk-1 phosphorylation is transmitted via FAK/Src and MAPKs pathways. Furthermore, confocal analysis clearly shows that FAK and the phosphorylated FAK at Y397 are localized in the nucleus and cytoplasm, whereas, the phosphorylated Elk-1-like protein is exclusively localized in the nucleus. Finally, co-immunoprecipitation and reciprocal co-immunoprecipitation analysis demonstrated the association of low molecular weight protein bands recognized by FAK antibodies, with Elk-1 or phospho-Elk-1 at ser 383 and confocal microscopy specifies that this association occurs only in the nucleus. These results are strongly supporting that Elk-1-like protein is a novel protein-binding partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk-1 generally. Evidently, the complex participates in the process of phagocytosis in medfly hemocytes.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Carotenoids in evolutionary ecology: re‐evaluating the antioxidant role

The antioxidant role of carotenoids in the living organism was proposed as a possible basis for the honesty of carotenoid‐based signals. However, recent studies have questioned the relevance of carotenoids as powerful antioxidants in vivo. Current evidence does not seem to support the “antioxidant role” hypothesis, but it does not allow us to reject it either. This paper proposes some steps to solve this controversy, such as taking a dynamic approach to antioxidant responses, designing protocols that expose individuals to oxidative challenges, analyzing tissues other than blood, and obtaining measures of antioxidant capacity and oxidative damage simultaneously. However, it should be considered that, irrespective of their antioxidant potential, carotenoids might still give information on oxidative stress levels if they are particularly sensitive to free radicals. Finally, lumping together the immunostimulatory and antioxidant roles of carotenoids should be avoided as these functions are not necessarily associated.     

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.     

Loss of Notch2 and Notch3 in vascular smooth muscle causes patent ductus arteriosus

The overlapping roles of the predominant Notch receptors in vascular smooth muscle cells, Notch2 and Notch3, have not been clearly defined in vivo. In this study, we use a smooth muscle‐specific deletion of Notch2 together with a global Notch3 deletion to produce mice with combinations of mutant and wild‐type Notch2/3 alleles in vascular smooth muscle cells. Mice with complete loss of Notch3 and smooth muscle‐expressed Notch2 display late embryonic lethality and subcutaneous hemorrhage. Mice without smooth muscle‐Notch2 and only one wild‐type copy of Notch3 die within one day of birth and present with vascular defects, most notably patent ductus arteriosus (DA) and aortic dilation. These defects were associated with decreased expression of contractile markers in both the DA and aorta. These results demonstrate that Notch2 and Notch3 have overlapping roles in promoting development of vascular smooth muscle cells, and together contribute to functional closure of the DA. genesis 53:738–748, 2015. © 2015 Wiley Periodicals, Inc.     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.     

Losartan inhibits endothelial-to-mesenchymal transformation in mitral valve endothelial cells by blocking transforming growth factor-β-induced phosphorylation of ERK

Adult cardiac valve endothelial cells (VEC) undergo endothelial to mesenchymal transformation (EndMT) in response to transforming growth factor-β (TGFβ). EndMT has been proposed as a mechanism to replenish interstitial cells that reside within the leaflets and further, as an adaptive response that increases the size of mitral valve leaflets after myocardial infarction. To better understand valvular EndMT, we investigated TGFβ-induced signaling in mitral VEC, and carotid artery endothelial cells (CAEC) as a control. Expression of EndMT target genes α-smooth muscle actin (α-SMA), Snai1, Slug, and MMP-2 were used to monitor EndMT. We show that TGFβ-induced EndMT increases phosphorylation of ERK (p-ERK), and this is blocked by Losartan, an FDA-approved antagonist of the angiotensin II type 1 receptor (AT1), that is known to indirectly inhibit phosphorylation of ERK (p-ERK). Blocking TGF-β-induced p-ERK directly with the MEK1/2 inhibitor RDEA119 was sufficient to prevent EndMT. In mitral VECs, TGFβ had only modest effects on phosphorylation of the canonical TGF-β signaling mediator mothers against decapentaplegic homolog 3 (SMAD3). These results indicate a predominance of the non-canonical p-ERK pathway in TGFβ-mediated EndMT in mitral VECs. AT1 and angiotensin II type 2 (AT2) were detected in mitral VEC, and high concentrations of angiotensin II (AngII) stimulated EndMT, which was blocked by Losartan. The ability of Losartan or MEK1/2 inhibitors to block EndMT suggests these drugs may be useful in manipulating EndMT to prevent excessive growth and fibrosis that occurs in the leaflets after myocardial infarction.