Calcium-dependent Protein Kinase Activity Research Articles

Visualizing calcium-dependent signaling networks in plants

Calcium-dependent protein kinases (CDPKs) are a multigene protein kinase family that have key regulatory roles in plants. However, imaging CDPK signals in plant cells remains challenging. The recently developed genetically encoded CDPK-Förster resonance energy transfer (FRET) reporter developed by Liese et al. allows visualization of calcium (Ca2+)-dependent conformational changes during activation or inactivation of CDPKs, providing a powerful tool for real-time monitoring of calcium decoding in plants.

Pathogen Xanthomonas campestris-induced immune-related genes integrated with phytohormones are involved in stomatal closure by interactively regulating ROS and Ca2+ signaling in the Xcc–Brassica napus pathosystem

This study aimed to characterize Xanthomonas campestris pv. campestris (Xcc)-induced ABA interaction with immune-related genes in stomatal movement. Xcc-responsive alterations in resistance genes, Ca2+, reactive oxidative species, and phytohormones were compared with those of exogenous ABA application (Exo-ABA) as being linked to stomatal movement. Xcc infection up-regulated botrytis-induced kinase 1 (BIK1) with a significant increase in salicylic acid (SA) and jasmonic acid (JA) levels at the early phase of infection (2 days after post-inoculation; DPI), this treatment also increased TIR-NB-LRR-type R-gene (TAO1) and R-gene–mediated signaling gene (SGT1) expression as well as SA and ABA accumulation during the later phase (7–15 DPI). In contrast, the Exo-ABA treatment did not activate these genes, even though the ABA-receptor gene PYR1 was up-regulated. The accumulation of Ca2+ and H2O2 in Xcc-inoculated leaves resulted from the activation of calcium-dependent protein kinase 5 (CDPK5), SA synthesis and signaling genes (ICS1 and NPR1), and ABA-related genes (NCED3 and MYC2), while in Exo-ABA treated leaves via the activation of H2O2-related genes (NADPH oxidase and OXI1) and calmodulin (CaM) in predominant ABA responses, leading to a depression of SA responses. Furthermore, the expression of immune-responsive mitogen-activated protein kinase 3 (MAPK3) was prominent in Xcc-inoculated leaves, while MAPK9 was more prominently expressed in Exo-ABA-treated leaves. Both Xcc infection and Exo-ABA treatments led to stomatal closure, accompanied by enhanced expression of slow anion channel 1 (SLAC1). These results indicate that Xcc-induced BIK1-SA-JA interaction acts as pattern-triggered immunity (PTI) in the early phase, and then after which TAO1-SA-ABA mediates CDPK5-dependent Ca2+ and H2O2 generation, leading to MAPK3-SLAC1-mediated stomatal closure in the later phase, while in Exo-ABA-treated leaves via ABA-mediated CaM-dependent H2O2 accumulation and stomata closure.

Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway.

Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (μ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2-20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

Extracellular Histone-Induced Protein Kinase C Alpha Activation and Troponin Phosphorylation Is a Potential Mechanism of Cardiac Contractility Depression in Sepsis.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and βII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCβII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCβII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.

Characteristics analyses of Eimeria tenella 14-3-3 protein and verification of its interaction with calcium-dependent protein kinase 4

Avian coccidiosis is a common disease caused by Eimeria spp. In the genus Eimeria, the species Eimeria tenella is an obligate intracellular parasite that invades mostly chicken cecal epithelial cells. The 14-3-3 protein is one of the most common adaptor proteins. It is involved in regulating protein phosphorylation and is associated with phosphorylated proteins to regulate signal transduction. Previous reports have shown that 14-3-3 protein has a direct regulatory effect on calcium-dependent protein kinases (CDPKs) activity by interacting with CDPKs. In this study, the characteristics of the E. tenella 14-3-3 protein including transcription and translation analyses, localization in different developmental stages etc were analyzed. The interaction between E. tenella 14-3-3 (Et14-3-3) and E. tenella calcium-dependent protein kinase 4 (EtCDPK4) which is a critical molecule in E. tenella invasion of host cells was verified by Bimolecular Fluorescent Complimentary (BiFC), Co-Immunoprecipitation (co-IP), and Glutathione S-transferase (GST) pull-down. The transcription and translation levels were analyzed using real-time quantitative PCR and western blot. The results showed that the mRNA transcription level of Et14-3-3 was highest in the sporozoite, and the translation level was higher in the unsporulated oocyst than in the other stages. Indirect immunolocalization found that Et14-3-3 was located mainly at the anterior of sporozoites and on the surface of second-generation merozoites. As the sporozoites developed in cells, the fluorescence intensity of Et14-3-3 gradually darkened. BiFC results showed green fluorescence under microscopy in 293T cells co-transfected with pBiFC-VN155-Et14-3-3 and pBiFC-VC155-EtCDPK4. Co-IP and GST pull-down showed that Et14-3-3 interacted with EtCDPK4, which is consistent with the BiFC results. These results indicated that Et14-3-3 had significant interactions with EtCDPK4. Co-localization of Et14-3-3 with EtCDPK4 in sporozoites revealed that they were located in the same position. The secretion assay results indicated that Et14-3-3 was a secreted protein but was not secreted from micronemes. These results lay the foundation for further research on the mechanism of action of EtCDPK4 with Et14-3-3 and the functions of Et14-3-3 in the lifecycle of E. tenella.

Plant Low-Temperature Stress: Signaling and Response

Cold stress has always been a significant limitation for plant development and causes substantial decreases in crop yield. Some temperate plants, such as Arabidopsis, have the ability to carry out internal adjustment, which maintains and checks the metabolic machinery during cold temperatures. This cold acclimation process requires prior exposure to low, chilling temperatures to prevent damage during subsequent freezing stress and maintain the overall wellbeing of the plant despite the low-temperature conditions. In comparison, plants of tropical and subtropical origins, such as rice, are sensitive to chilling stress and respond differently to low-temperature stress. Plants have evolved various physiological, biochemical, and molecular mechanisms to sense and respond to low-temperature stress, including membrane modifications and cytoskeletal rearrangement. Moreover, the transient increase in cytosolic calcium level leads to the activation of many calcium-binding proteins and calcium-dependent protein kinases during low-temperature stress. Recently, mitogen-activated protein kinases have been found to regulate low-temperature signaling through ICE1. Besides, epigenetic control plays a crucial role during the cold stress response. This review primarily focuses on low-temperature stress experienced by plants and their strategies to overcome it. We have also reviewed recent progress and previous knowledge for a better understanding of plant cold stress response.

Cadmium Accumulation Involves Synthesis of Glutathione and Phytochelatins, and Activation of CDPK, CaMK, CBLPK, and MAPK Signaling Pathways in Ulva compressa.

In order to analyze the effect of cadmium in Ulva compressa (Chlorophyta), the alga was cultivated with 10, 25, and 50 μM of cadmium for 7 days, and the level of intracellular cadmium was determined. Intracellular cadmium showed an increase on day 1, no change until day 5, and an increase on day 7. Then, the alga was cultivated with 10 μM for 7 days, and the level of hydrogen peroxide, superoxide anions, and lipoperoxides; activities of antioxidant enzymes ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); the level of glutathione (GSH) and ascorbate (ASC); and the level of phytochelatins (PCs) and transcripts encoding metallothioneins (UcMTs) levels were determined. The level of hydrogen peroxide increased at 2 and 12 h, superoxide anions on day 1, and lipoperoxides on days 3 to 5. The activities of AP and GR were increased, but not the DHAR activity. The level of GSH increased on day 1, decreased on day 3, and increased again on day 5, whereas ASC slightly increased on days 3 and 7, and activities of enzymes involved in GSH and ASC synthesis were increased on days 3 to 7. The level of PC2 and PC4 decreased on day 3 but increased again on day 5. The level of transcripts encoding UcMT1 and UcMT2 increased on days 3 to 5, mainly that of UcMT2. Thus, cadmium accumulation induced an oxidative stress condition that was mitigated by the activation of antioxidant enzymes and synthesis of GSH and ASC. Then, the alga cultivated with inhibitors of calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calcineurin B-like protein kinases (CBLPKs), and MAPKs and 10 μM of cadmium for 5 days showed a decrease in intracellular cadmium and in the level of GSH and PCs, with the four inhibitors, and in the level of transcripts encoding UcMTs, with two inhibitors. Thus, CDPKs, CaMK, CBLPKS, and MAPKs are involved in cadmium accumulation and GSH and PC synthesis, and GSH and PCs and/or UcMTs may participate in cadmium accumulation.

Role of copper in the enhancement of astaxanthin and lipid coaccumulation in Haematococcus pluvialis exposed to abiotic stress conditions

This study investigated the effects of copper (Cu) on astaxanthin and lipid biological synthesis in unicellular alga Haematococcus pluvialis under high-light (HL) and nitrogen-deficiency (ND) conditions. During a 15-day cultivation period, the astaxanthin and lipid contents reached the peak values (3.32% and 47.72%) under 6 μM Cu treatment, which were increased by 66.87% and 34.99% compared to nontreated group, respectively. The application of Cu also increased the transcriptional expression of biosynthesis genes and antioxidant enzyme-related genes, as well as increased the intracellular calcium (Ca2+) level but led to a decrease in reactive oxygen species (ROS) levels. Additionally, Cu treatment induced the activation of calcium-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs). This approach simultaneously facilitated astaxanthin and lipid production, and the role of Cu were elucidated on the regulation of signal transduction (e.g., Ca2+, CDPK, MAPK and ROS) in the carotenogenesis and lipogenesis in H. pluvialis.

Arabidopsis KASH Proteins SINE1 and SINE2 Are Involved in Microtubule Reorganization During ABA-Induced Stomatal Closure.

Abscisic acid (ABA) induces stomatal closure by utilizing complex signaling mechanisms, allowing for sessile plants to respond rapidly to ever-changing environmental conditions. ABA regulates the activity of plasma membrane ion channels and calcium-dependent protein kinases, Ca2+ oscillations, and reactive oxygen species (ROS) concentrations. Throughout ABA-induced stomatal closure, the cytoskeleton undergoes dramatic changes that appear important for efficient closure. However, the precise role of this cytoskeletal reorganization in stomatal closure and the nature of its regulation are unknown. We have recently shown that the plant KASH proteins SINE1 and SINE2 are connected to actin organization during ABA-induced stomatal closure but their role in microtubule (MT) organization remains to be investigated. We show here that depolymerizing MTs using oryzalin can restore ABA-induced stomatal closure deficits in sine1-1 and sine2-1 mutants. GFP-MAP4-visualized MT organization is compromised in sine1-1 and sine2-1 mutants during ABA-induced stomatal closure. Loss of SINE1 or SINE2 results in loss of radially organized MT patterning in open guard cells, aberrant MT organization during stomatal closure, and an overall decrease in the number of MT filaments or bundles. Thus, SINE1 and SINE2 are necessary for establishing MT patterning and mediating changes in MT rearrangement, which is required for ABA-induced stomatal closure.

Effects of Penicillium infection on the expression and activity of CDPK2 in postharvest Hami melon treated with calcium chloride

Calcium ions are second messengers in plant signal transduction involved in plant growth and development and the response to stress. Calcium-dependent protein kinase (CDPK) is calcium ion receptor functioning in plant disease resistance. Herein, we investigated changes in CDPK activity and resistance to Penicillium in postharvest Hami melons after different treatments. The non-infection group was permeated with calcium chloride (CaCl2) solution, while the infection group was permeated with CaCl2 solution and infected with Penicillium. CDPK activity was elevated in both groups but was higher in the infection group. Analysis of the molecular mechanism of CPDK in Penicillium infection revealed the 1635-bp HmCDPK2 gene with an open reading frame of 1239 nucleotides, encoding a protein of 412 amino acids with a molecular weight of 46161.61 Da and a theoretical isoelectric point of 5.00. The HmCDPK2 protein shares 40.11% tertiary structure similarity with CDPK from the apicomplexan parasite Plasmodium Bergheii (PDB ID 3Q5I). Fluorescence quantitative PCR results showed that expression of HmCDPK2 was upregulated in the infection group relative to the non-infection group, peaking at 12 h after treatment. We conclude that Hami melon develops resistance to Penicillium infection by regulating CDPK activity and upregulating HmCDPK2 expression.

Induction of calcium-dependent protein kinase activity and HmCDPK1 expression in the early response of Hami melons to Penicillium infection

Calcium-dependent protein kinase (CDPK) plays an important role in plant resistance to disease. In this study, we assessed CDPK activity in Hami melons during Penicillium infection, in order to investigate the possible role of CDPK in this context. The results showed the induction of CDPK by Penicillium infection may contribute to the early stages of disease resistance in Hami melons. In order to further study the molecular mechanisms underlying this response, relatively high expression of HmCDPK1 was screened form the transcriptome database of Hami melons. Bioinformatics analysis showed HmCDPK1 had a length of 2365 bp, with a maximum open reading frame of 1560 nucleotides, encoding 519 amino acids with a molecular weight of 58,646.83. Moreover, its theoretical isoelectric point was 6.34, as a hydrophilic and non-transmembrane protein. Real-time quantitative PCR showed the transcript of HmCDPK1 was persistently increased after Penicillium infection and reached its maximum at 12 h, being significantly higher than in non-infected plants. These results suggest the induction of CDPK activity and HmCDPK1 expression in the early response of Hami melons to Penicillium may contribute to the resistance against this infection.

Constitutive activation of calcium-dependent protein kinase 3 confers a drought tolerance by inhibiting inward K+ channel KAT1 and stomatal opening in Arabidopsis

Constitutive activation of calcium-dependent protein kinase 3 confers a drought tolerance by inhibiting inward K+ channel KAT1 and stomatal opening in Arabidopsis

A calcium-dependent protein kinase, ZmCPK32, specifically expressed in maize pollen to regulate pollen tube growth.

Calcium-dependent protein kinases (CPKs) play an essential role in the regulation of pollen tube growth. Although CPK genes have been identified in maize, and some have been functionally characterized, the molecular function of ZmCPKs associated with pollen tube development remains less well studied. Here, we report that a pollen-specific CPK, ZmCPK32, is involved in the regulation of pollen germination and tube extension. ZmCPK32 exhibited CPK activity and was localized on the plasma membrane and punctate internal membrane compartments via N-terminal acylation. In situ hybridization and real-time PCR revealed that ZmCPK32 transcripts accumulated in pollen and expression was dramatically upregulated during shedding. To elucidate the function of this gene, we transiently expressed a ZmCPK32-GFP fusion protein in tobacco pollen using microparticle bombardment. ZmCPK32 accumulation inhibited pollen germination and reduced pollen tube growth, but this effect was abolished when the kinase-inactive variant was expressed, indicating that kinase activity is critical for its regulatory function. In addition, the plasma membrane localization of ZmCPK32 is essential for regulating polar growth, as pollen expressing the cytosol-localized kinase displayed reduced tube length but germinated well. Moreover, the constitutively active form of ZmCPK32 enhanced the reduction in the germination rate, indicating that the specific activation of ZmCPK32 via calcium ions at the cortical growth point is essential for regulating appropriate germination. The results suggest that ZmCPK32 is functionally associated with pollen tube growth, and could represent a potential target for breeding male-sterile maize.

Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G.

ABSTRACTWe used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes.

Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.

Allosteric activation of apicomplexan calcium-dependent protein kinases

Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

Toward a Molecular Understanding of Adaptive Immunity: A Chronology, Part III.

Early reports on T cell antigen receptor (TCR) signaling uncovered a rapid increase in intracellular calcium concentration and the activation of calcium-dependent protein kinase as necessary for T cell activation. Cytolytic T cell clones were instrumental in the discovery of intracellular cytolytic granules, and the isolation of the perforin and granzyme molecules as the molecular effectors of cell-mediated lysis of target cells via apoptosis. Cytolytic T cell clones and TCR cDNA clones were also instrumental for the generation of TCR transgenic animals, which provided definitive evidence for negative selection of self-reactive immature thymocytes. In addition, studies of TCR complex signaling of immature thymocytes compared with mature T cells were consistent with the interpretation that negative selection occurs as a consequence of the incapacity of immature cells to produce IL-2, resulting in cytokine deprivation apoptosis. By comparison, taking advantage of cloned TCRs derived from T cell clones reactive with male-specific molecules, using TCR transgenic mice it was possible to document positive selection of female thymocytes when the male-specific molecules were absent. Focusing on the molecular mechanisms of T cell “help” for the generation of antibody-forming cells following the path opened by the elucidation of the IL-2 molecule, several groups were successful in the identification, isolation, and characterization of three new interleukin molecules (IL-4, IL-5, and IL-6) that promote the proliferation and differentiation of B cells. In addition, the identification of a B cell surface molecule (CD40) that augmented B cell antigen receptor-stimulated proliferation and differentiation led to the discovery of a T cell activation surface molecule that proved to be the CD40-ligand, thus finally providing a molecular explanation for “linked or cognate” recognition when T cells and B cells interact physically. Accordingly, the decade after the generation of the first T cell clones saw the elucidation of the molecular mechanisms of T cell cytotoxicity and T cell help, thereby expanding the number of molecules responsible for adaptive T cell immunity.

Metformin enhances glucagon-like peptide 1 via cooperation between insulin and Wnt signaling

One aspect of the effects of metformin on glucagon-like peptide (GLP)-1 might be associated with the mechanism by which the cross talk between insulin and Wnt signaling enhances GLP1 secretion, due to the action of metformin as an insulin sensitizer. However, this remains completely unknown. In this study, we have investigated the mechanisms of the action of metformin on cross talk between insulin and Wnt signaling. GLP1 enhancement by meformin was determined in human NCI-H716 intestinal L-cells and hyperglycemic db/db mice treated with metformin (0.25 and 0.5 mM and/or 12.5 mg/kg body weight) for 24 h and 2 months. Metformin increased GLP1 secretion in L-cells and db/db mice. Metformin stimulated the nuclear translocation of β-catenin and TOPflash reporter activity, and gene depletion of β-catenin or enhancement of mutation of transcription factor 7-like 2 binding site offset GLP1. In addition, insulin receptor substrate 2 gene depletion blocked metformin-enhanced β-catenin translocation. These effects were preceded by an increase in glucose utilization and calcium influx, the activation of calcium-dependent protein kinase, and, in turn, the activation of insulin signaling, and the inhibition of glycogen synthase kinase 3β, a potent inhibitor of β-catenin. Furthermore, high blood glucose levels were controlled via GLP1 receptor-dependent insulinotropic pathways in db/db mice, which were evidenced by the increase in GLP1 and insulin levels at 30 min after oral glucose loading and pancreatic insulinotropic gene expression. Our findings indicate that the cooperation between Wnt and its upstream insulin signaling pathways might be a novel and important mechanism underlying the effects of metformin on GLP1 production.

Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca2+/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.

Expression, purification and biochemical characterization of recombinant Ca-dependent protein kinase 2 of the malaria parasite Plasmodium falciparum

Calcium-dependent protein kinases (CDPKs) are serine/threonine kinases that react in response to calcium which functions as a trigger for several mechanisms in plants and invertebrates, but not in mammals. Recent structural studies have defined the role of calcium in the activation of CDPKs and have elucidated the important structural changes caused by calcium in order to allow the kinase domain of CDPK to bind and phosphorylate the substrate. However, the role of autophosphorylation in CDPKs is still not fully understood. In Plasmodium falciparum, seven CDPKs have been identified by sequence comparison, and four of them have been characterized and assigned to play a role in parasite motility, gametogenesis and egress from red blood cells. Although PfCDPK2 was already discovered in 1997, little is known about this enzyme and its metabolic role. In this work, we have expressed and purified PfCDPK2 at high purity in its unphosphorylated form and characterized its biochemical properties. Moreover, propositions about putative substrates in P. falciparum are made based on the analysis of the phosphorylation sites on the artificial substrate myelin basic protein (MBP).