Calcium-calmodulin Dependent Kinase Research Articles

Phosphorylation of Myosin Increases the ATPase Activity of Relaxed Skeletal Muscle Fibers

Myosin in skeletal muscle is phosphorylated by a calcium-calmodulin dependent kinase during periods of sustained activity. Phosphorylation of myosin is known to destabilize the array of myosin heads bound to the core of the thick filament in relaxed muscle, known as the interacting heads motif. Myosin heads in this array are associated with a state with a highly inhibited ATPase activity, known as the Super Relaxed State (SRX). To determine whether phosphorylation of myosin increases the ATPase activity, we measured the ATPase activity of relaxed, skinned skeletal fibers in the presence and absence of myosin phosphorylation. Fibers were phosphorylated, >50%, by including phosphatase inhibitors in the glycerinating solution. Control fibers had an ATPase activity of 0.040 ± 0.004 s−1. Phosphorylated fibers had an ATPase activity of 0.060 ± 0.002 s−1, a 50 % increase. We have recently found a small molecule, piperine that destabilizes the SRX, increasing relaxed fiber ATPase activity. In the presence of 100μM piperine the control fibers had an ATPase activity of 0.064 ± 0.004 s−1, the phosphorylated fibers had an ATPase activity of 0.057 ± 0.001 s−1. Thus both phosphorylation and piperine increase the ATPase activity of resting fibers by destabilizing a fraction of myosin heads in the SRX, 30-50%, but their effects are not additive, suggesting that they are each affecting the same population of myosin heads. One candidate for this population may be the “free” head of the interacting heads motif, which is known to be less stable than the other head. As myosin remains in a phosphorylated state for several minutes following a period of activity, the effect described above will contribute to the energetic cost of physical activity, particularly during light activity.

Cellular subtype expression and activation of CaMKII regulate the fate of atherosclerotic plaque

Background and aimsAtherosclerosis is a degenerative process of the arterial wall implicating activation of macrophages and proliferation of vascular smooth muscle cells. Calcium-calmodulin dependent kinase type II (CaMKII) in vascular smooth muscle cells (VSMCs) regulates proliferation, while in macrophages, this kinase governs diapedesis, infiltration and release of extracellular matrix enzymes. We aimed at understanding the possible role of CaMKII in atherosclerosis plaques to regulate plaque evolution towards stability or instability. MethodsClinically defined stable and unstable plaques obtained from patients undergoing carotid end arteriectomy were processed for evaluation of CaMKs protein expression, activity and localization. ResultsThe larger content of CaMKII was found in CD14+myeloid cells that were more abundant in unstable rather than stable plaques. To test the biological effect of activated CD14+myeloid cells, VSMCs were exposed to the conditioned medium (CM) of macrophages extracted from carotid plaques. CM induced attenuation of CaMKs expression and activity in VSMCs, leading to the reduction of VSMCs proliferation. This appears to be due to the CaMKII dependent release of cytokines. ConclusionsThese results indicate a pivotal role of CaMKs in atherosclerosis by regulating activated myeloid cells on VSMCs activity. CaMKII could represent a possible target for therapeutic strategies based on macrophages specific inhibition for the stabilization of arteriosclerotic lesions.

Emodin alleviates hepatic steatosis by inhibiting sterol regulatory element binding protein 1 activity by way of the calcium/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mechanistic target of rapamycin-p70 ribosomal S6 kinase signaling pathway.

To investigate the effects of emodin on the treatment of non-alcoholic fatty liver and the underlying mechanisms. In vitro, hepatocytes were treated with 1mM free fatty acid together with various concentrations of emodin. In vivo, Sprague-Dawley rats were divided into a control group, high-fat diet (HFD) group, and three HFD groups treated with 40, 80, and 160mg/kg emodin, respectively. After being fed a HFD for 4weeks, rats were orally dosed with emodin once daily for 8weeks. The biochemical parameters and histology features were examined. The expression of lipogenic and lipolytic gene and protein and the phosphorylation of calcium/calmodulin-dependent kinase kinase (CaMKK), AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR) and p70 ribosomal S6 kinase (p70S6K) were measured in vitro and in vivo. Emodin improved lipid accumulation in vitro and in vivo. Emodin downregulated the levels of sterol regulatory element binding protein 1 (SREBP1) and its target enzymes but increased lipolysis-related proteins and mRNA. Phosphorylation of AMPK was increased, while phosphorylation of mTOR and p70S6K were suppressed by emodin. The nuclear translocation of SREBP1 was inhibited by emodin by AMPK and mTOR. Emodin activated AMPK by CaMKK and reversed the reduction of CaMKK in HFD-fed rats. Emodin effectively ameliorates hepatic steatosis through the CaMKK-AMPK-mTOR-p70S6K-SREBP1 signaling pathway.

Protein kinase C gamma-mediated phosphorylation of GluA1 in the postsynaptic density of spinal dorsal horn neurons accompanies neuropathic pain, and dephosphorylation by calcineurin is associated with prolonged analgesia.

Loss of calcineurin (protein phosphatase 3) activity and protein content in the postsynaptic density (PSD) of spinal dorsal horn neurons was associated with pain behavior after chronic constriction injury (CCI) of the rat sciatic nerve, and intrathecal administration of the phosphatase provided prolonged analgesia (Miletic et al. 2013). In this study, we examined whether one consequence of the loss of calcineurin was the persistent phosphorylation of the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPAR) receptors in the PSD. This would allow continual activation of AMPAR receptors at the synapse to help maintain a long-lasting enhancement of synaptic function, ie, neuropathic pain. We also investigated if the phosphorylation was mediated by protein kinase A (PKA), protein kinase C gamma (PKCγ), or calcium-calmodulin dependent kinase II (CaMKII), and if the prolonged calcineurin analgesia was associated with GluA1 dephosphorylation. Mechanical thresholds and thermal latencies were obtained before CCI. Seven days later, the behavioral testing was repeated before saline, calcineurin, or the specific peptide inhibitors of PKA (PKI-tide), PKCγ (PKC 19-31), or CaMKII (autocamtide-2-related inhibitory peptide) were injected intrathecally. The behavior was retested before the animals were euthanized and their PSD isolated. All CCI animals developed mechanical and thermal hypersensitivity. This was associated with phosphorylation of GluA1 in the ipsilateral PSD at Ser831 (but not Ser845) by PKCγ and not by PKA or CaMKII. Intrathecal treatment with calcineurin provided prolonged analgesia, and this was accompanied by GluA1 dephosphorylation. Therapy with calcineurin may prove useful in the prolonged clinical management of well-established neuropathic pain.

Abstract 242: A Novel Transient Receptor Potential Ion Channel Vanilloid 4 Agonist Inhibits Monocytes Adhesion and Atherosclerosis via eNOS Activation

Endothelial nitric oxide (NO) synthase (eNOS/NOS3)-derived NO represents an important vasculoprotective molecule in the cardiovascular system. The transient receptor potential ion channel vanilloid subtype 4 (TRPV4) mediates vascular mechanosensitivity, in part through an increase in blood flow-mediated NO production and endothelium-dependent relaxation. Here, we hypothesized that non-mechanical TRPV4 activation can mimic laminar flow-induced signaling and thereby limits monocyte adhesion and atherosclerosis. We observed that GSK1016790A, a novel, potent and specific agonist of TRPV4, mimics laminar flow, thereby inducing robust phosphorylation (Ser1177) of eNOS in a TRPV4 and calcium-dependent manner. Mechanistically, GSK1016790A induced eNOS phosphorylation, NO production and vasorelaxation via calcium/calmodulin-dependent kinase kinase (CaMKK)/AMP-activated protein kinase (AMPK) pathway. In functional experiments, GSK1016790A inhibited TNFα-induced monocytes adhesion to human endothelial cells by inhibiting the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), and this inhibition was abolished by an eNOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) or AMPK inhibitor Compound C. Mice given GSK1016790A showed increased eNOS (2.5- fold increase versus vehicle, n=12, P<0.001) and AMPK phosphorylation (2.1- fold increase versus vehicle, n=12, P<0.01) in the aorta. Finally, we demonstrated that oral administration of GSK1016790A inhibited atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet (13.27% ± 1.571%, versus vehicle 23.22% ± 1.130%, n=8, P<0.001). In conclusion, our data provide a novel mechanism whereby a TRPV4 agonist triggers NO production and suggests that TRPV4 agonists could be used as innovative vasculoprotective treatments of endothelial dysfunction-related diseases, such as atherosclerosis, hypertension and stroke.

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

Inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with IP3 (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.

Intracellular translocation of histone deacetylase 5 regulates neuronal cell apoptosis

Histone deacetylase 5 (HDAC5) undergoes signal-dependent shuttling between the nucleus and cytoplasm, which is regulated in part by calcium/calmodulin-dependent kinase (CaMK)-mediated phosphorylation. Here, we report that HDAC5 regulates the survival of cortical neurons in pathological conditions. HDAC5 was evenly localized to the nucleus and cytoplasm in cultured cortical neurons. However, in response to 50μM NMDA conditions that induced neuronal cell apoptosis, nuclear-distributed HDAC5 was rapidly phosphorylated and translocated into cytoplasm of the cultured cortical neurons. Treatment with a CaMKII inhibitor KN93 suppressed HDAC5 phosphorylation and nuclear translocation induced by NMDA, whereas constitutively active CaMKIIα (T286D) stimulated HDAC5 nuclear export. Importantly, we showed that ectopic expression of nuclear-localized HDAC5 in cortical neurons suppressed NMDA-induced apoptosis. Finally, inactivation of HDAC5 by treatment with the class II-specific HDAC inhibitor trichostatin A (TSA) promoted NMDA-induced neuronal cell apoptosis. Altogether, these data identify HDAC5 and its intracellular translocation as key effectors of multiple pathways that regulate neuronal cell apoptosis.

Calcium Calmodulin Dependent Kinase Kinase 2 Regulates Hematopoietic Stem Cell Regeneration and Quiescence

Extracellular free calcium (Ca2+) is the most abundant signaling molecule in the hematopoietic stem cell (HSC) niche, and intracellular calcium is an important second messenger controlling critical functions in HSCs. Accordingly, Ca2+ is a master regulator of HSC biology, and nodes of the Ca2+ signaling network are emerging as critical components of the molecular machinery governing HSC regeneration and quiescence. Ca2+ controls many cell functions by forming a complex with Calmodulin (CaM), which binds and activates a family of Calcium/Calmodulin dependent kinase (CaMK) that includes CaMKI, CaMKII, CaMKIV, CaMKK1 and CaMKK2. We have previously investigated the function of CaMKK2 in blood cells and established this protein as a critical component of the calcium signaling pathway that regulates granulopoiesis and macrophage activation. Here, we investigated the function of CaMKK2 in the regenerative response of HSCs. To determine the expression of CaMKK2 under homeostatic conditions, we examined transgenic reporter mice and identified robust reporter activity in primitive long-term hematopoietic stem cells, but reduced signal in the majority of lineage positive blood and bone marrow cells. To establish whether CaMKK2 has a functional role in controlling hematopoiesis and HSC self-renewal, we examined hematopoietic regeneration in mice deficient for CaMKK2. We demonstrate that Camkk2 -/- mice have accelerated bone marrow and peripheral blood regeneration following hematopoietic injury. Camkk2 -/- HSCs showed increased proliferative capacity in vivo and mobilized into the peripheral blood to establish extra-medullary hematopoiesis during regeneration. To identify the proximal target of CaMKK2, we assessed the phosphorylation status of CaMKI, CaMKIV and adenosine monophosphate activated protein kinase (AMPK), which are known targets of CaMKK2. Our results indicate Camkk2 -/- stem and progenitor cells (HSPC) fail to accumulate phospho-AMPK in response to ionizing radiation. Since AMPK is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, we also analyzed the levels of phospho-ribosomal 6 protein (rpS6), which is a distal target of the mTOR cascade that positively regulates cell size and proliferation. We found that irradiated Camkk2 -/- HSPCs accumulate higher levels of phospho-rpS6 compared to wild type cells. This finding implicates CaMKK2 as a critical component of a negative regulatory circuit that links calcium signaling to AMPK activation and cell proliferation in HSPC. Cumulatively, our data demonstrate that CaMKK2 regulates HSC function and suggests CaMKK2 inhibition is a novel approach to stem cell expansion for clinical therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 19320: Protein Kinase G Oxidation Blunts Its Suppression of Cardiac Hypertrophy by Diminishing Phosphorylation of Tuberin and Consequent Autophagy

Stimulation of protein kinase G (PKG) suppresses maladaptive cardiac hypertrophy in part by blunting signaling cascades such as calcineurin/NFAT (Cn) and calcium-calmodulin dependent kinase II (CamKII). Here we report PKG stimulation also enhances autophagy as an anti-hypertrophic mechanism, identify a novel phosphorylation target for this effect, and show this signaling is blunted when PKG becomes oxidized at cysteine 42 (C42). Mice with knock-in expression of mutant PKG (C42S) or littermate controls were subjected to pressure-overload. C42S mice were protected, with reduced hypertrophy and fibrosis, improved function, and blunted Cn, CamKII, and hypertrophic gene activation. Neonatal rat ventricular myocytes (NRVMs) expressing C42S-PKG exposed to 48hr endothelin-1 (ET1) also displayed less hypertrophic responses. In both C42S mice and NRVMs expressing C42S, autophagic flux was increased over WT, determined by autophagic flux assays, which was associated with inhibited mammalian target of rapamycin (mTOR) signaling. Control mice improved responses similar to C42S mice if autophagic flux was stimulated by inhibiting mTOR with everolimus. Analogous findings were obtained by mTOR suppression in NRVMs. To determine how PKG activation influences mTOR-regulated autophagy, we performed unbiased phospho-proteome analysis using mass spectrometry that revealed a unique PKG targeted site on tuberin (TSC2) at S1343. To test the functional importance of this site, we expressed a mutated TSC2, serine to glutamic acid (phospho-mimetic, S1343E), which depressed mTOR signaling, increased autophagic flux, and blunted hypertrophic signaling and cell enlargement stimulated by ET1 in NRVMs. By contrast, the cardio-protection elicited by PKG C42S was lost when TSC2 or a downstream critical autophagy protein, ATG5 were silenced. Taken together, these results provide the first evidence that PKG activation augments autophagy via targeting of TSC2 as a mechanism of anti-hypertrophic signaling, and that this occurs much more when PKG is in a reduced state (e.g. no C42-disulfide). Loss of this mechanism in myocardium under oxidative stress may play a role in the decline in protection from PKG in cardiac disease which often involves an oxidative environment.

PTK2b function during fertilization of the mouse oocyte

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

CaMKII Actomyosin Interactions in Live Cells

Synaptic stimulation leads to expansion of the dendritic spine caused by rearrangement of the actin cytoskeleton triggered by phosphorylation of the calcium calmodulin dependent kinase (CaMKII) and by myosin II activity. To study CaMKII-actomyosin interactions we have visualized single GFP-tagged CaMKII holoenzymes bound to tagRFP-tagged cortical actin in human endothelial cells by total internal reflection fluorescence microscopy. CaMKII phosphorylation mutants were used to examine whether release of CaMKII sequestered actin and myosin II induced F-actin fragmentation can explain the timing and extent of spine expansion. Thus far, we have found (i) Single GFP-CaMKII holoenzymes were resolved; in contrast to the less intense and rapidly mobile single GFP molecules. (ii) The holoenzymes have unimodal intensity distributions with < 30% standard deviation. The constitutively-active CaMKII T286D mutant, the sole exception, gave bimodal distributions with the higher mode six times as intense as the lower one. Subunit stoichiometries were determined by comparison against immobilized GFP single molecule intensities. (iii) CaMKII dwell-time distributions on stress fibers and filopodial structures were characterized. Dissociation rates were also measured by evanescent irradiation of photo-activatable GFP-CaMKII constructs bound to cortical actin (iv) The green / red fluorescence ratio of the actin structures relative to background gave an equilibrium measure of CaMKII binding. Affinity differences for different CaMKII isoforms and different phosphorylation states were estimated from the fluorescence ratios. In addition, single particle tracking was used to measure transient binding to the actin cytoskeleton. (v) We are currently measuring spatial co-operativity in the F-actin CaMKII binding sites and extending the methodology to analyse CaMKII diffusion along dendrites and spines in hippocampal neuron cultures. Our single molecule imaging data reveal spatial and macromolecular heterogeneities that may underlie aspects of the signal transduction response and cytoskeletal dynamics that are important for synaptic plasticity.

Forced running exercise attenuates hippocampal neurogenesis impairment and the neurocognitive deficits induced by whole-brain irradiation via the BDNF-mediated pathway

Cranial radiotherapy induces progressive and debilitating cognitive deficits, particularly in long-term cancer survivors, which may in part be caused by the reduction of hippocampal neurogenesis. Previous studies suggested that voluntary exercise can reduce the cognitive impairment caused by radiation therapy. However, there is no study on the effect of forced wheel exercise and little is known about the molecular mechanisms mediating the effect of exercise. In the present study, we investigated whether the forced running exercise after irradiation had the protective effects of the radiation-induced cognitive impairment. Sixty-four Male Sprague–Dawley rats received a single dose of 20Gy or sham whole-brain irradiation (WBI), behavioral test was evaluated using open field test and Morris water maze at 2months after irradiation. Half of the rats accepted a 3-week forced running exercise before the behavior detection. Immunofluorescence was used to evaluate the changes in hippocampal neurogenesis and Western blotting was used to assess changes in the levels of mature brain-derived neurotrophic factor (BDNF), phosphorylated tyrosine receptor kinase B (TrkB) receptor, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), calcium-calmodulin dependent kinase (CaMKII), cAMP-calcium response element binding protein (CREB) in the BDNF–pCREB signaling. We found forced running exercise significantly prevented radiation-induced cognitive deficits, ameliorated the impairment of hippocampal neurogenesis and attenuated the down-regulation of these proteins. Moreover, exercise also increased behavioral performance, hippocampal neurogenesis and elevated BDNF–pCREB signaling in non-irradiation group. These results suggest that forced running exercise offers a potentially effective treatment for radiation-induced cognitive deficits.

Melatonin ameliorates cognitive impairment induced by sleep deprivation in rats: Role of oxidative stress, BDNF and CaMKII

Sleep deprivation (SD) has been shown to induce oxidative stress which causes cognitive impairment. Melatonin, an endogenous potent antioxidant, protects neurons from oxidative stress in many disease models. The present study investigated the effect of melatonin against SD-induced cognitive impairment and attempted to define the possible mechanisms involved. SD was induced in rats using modified multiple platform model. Melatonin (15mg/kg) was administered to the rats via intraperitoneal injection. The open field test and Morris water maze were used to evaluate cognitive ability. The cerebral cortex (CC) and hippocampus were dissected and homogenized. Nitric oxide (NO) and malondialdehyde (MDA) levels and the superoxide dismutase (SOD) enzyme activity of hippocampal and cortical tissues (10% wet weight per volume) were performed to determine the level of oxidative stress. The expression of brain-derived neurotrophic factor (BDNF) and calcium-calmodulin dependent kinase II (CaMKII) proteins in CC and hippocampus was assayed by means of immunohistochemistry. The results revealed that SD impairs cognitive ability, while melatonin treatment prevented these changes. In addition, melatonin reversed SD-induced changes in NO, MDA and SOD in both of the CC and hippocampus. The results of immunoreactivity showed that SD decreased gray values of BDNF and CaMKII in CC and hippocamal CA1, CA3 and dentate gyrus regions, whereas melatonin improved the gray values. In conclusion, our results suggest that melatonin prevents cognitive impairment induced by SD. The possible mechanism may be attributed to its ability to reduce oxidative stress and increase the levels of CaMKII and BDNF in CC and hippocampus.

Signal Transduction in Cerebral Arteries after Subarachnoid Hemorrhage—A Phosphoproteomic Approach

After subarachnoid hemorrhage (SAH), pathologic changes in cerebral arteries contribute to delayed cerebral ischemia and poor outcome. We hypothesize such changes are triggered by early intracellular signals, targeting of which may prevent SAH-induced vasculopathy. We performed an unbiased quantitative analysis of early SAH-induced phosphorylations in cerebral arteries and evaluated identified signaling components as targets for prevention of delayed vasculopathy and ischemia. Labeled phosphopeptides from rat cerebral arteries were quantified by high-resolution tandem mass spectrometry. Selected SAH-induced phosphorylations were validated by immunoblotting and monitored over a 24-hour time course post SAH. Moreover, inhibition of key phosphoproteins was performed. Major SAH-induced phosphorylations were observed on focal adhesion complexes, extracellular regulated kinase 1/2 (ERK1/2), calcium calmodulin-dependent kinase II, signal transducer and activator of transcription (STAT3) and c-Jun, the latter two downstream of ERK1/2. Inhibition of ERK1/2 6-hour post SAH prevented increases in cerebrovascular constrictor receptors, matrix metalloprotease-9, wall thickness, and improved neurologic outcome. STAT3 inhibition partially mimicked these effects. The study shows that quantitative mass spectrometry is a strong approach to study in vivo vascular signaling. Moreover, it shows that targeting of ERK1/2 prevents delayed pathologic changes in cerebral arteries and improves outcome, and identifies SAH-induced signaling components downstream and upstream of ERK1/2.

Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium-calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC50 for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC50 values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC50 for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.

Role of the calcium-calmodulin dependent kinase 2 in medullary thyroid carcinoma

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Quantitative proteomics for identifying biomarkers for Rabies.

IntroductionRabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.MethodsIn this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.Results and conclusionWe identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.

S-Allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway

S-Allyl cysteine (SAC), a nontoxic garlic compound, has a variety of pharmacological properties, including antioxidant and hepatoprotective properties. In this report, we provide evidence that SAC prevented free fatty acid (FFA)-induced lipid accumulation and lipotoxicity in hepatocytes. SAC significantly reduced FFA-induced generation of reactive oxygen species, caspase activation and subsequent cell death. Also, SAC mitigated total cellular lipid and triglyceride accumulation in steatotic HepG2 cells. SAC significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Additionally, SAC down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and its target genes, including ACC and fatty acid synthase. Use of a specific inhibitor showed that SAC activated AMPK via calcium/calmodulin-dependent kinase kinase (CaMKK) and silent information regulator T1. Our results demonstrate that SAC activates AMPK through CaMKK and inhibits SREBP-1-mediated hepatic lipogenesis. Therefore, SAC has therapeutic potential for preventing nonalcoholic fatty liver disease.

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize topresynaptic nerve terminals and mediate key eventsincluding synaptogenesis and neurotransmission.While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle

The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle.