Calcium Calmodulin Research Articles

Secreted 14-3-3ε: discovery by proteomics of a novel biomarker and/or therapeutical target in osteoarthritis

Secreted 14-3-3ε: discovery by proteomics of a novel biomarker and/or therapeutical target in osteoarthritis

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.

β‐Arrestin‐kinase scaffolds: turn them on or turn them off?

G-protein-coupled receptors (GPCRs) can signal through heterotrimeric G-proteins or through β-arrestins to elicit responses to a plethora of extracellular stimuli. While the mechanisms underlying G-protein signaling is relatively well understood, the mechanisms by which β-arrestins regulate the diverse set of proteins with which they associate remain unclear. Multi-protein complexes are a common feature of β-arrestin-dependent signaling. The first two such complexes discovered were the mitogen-activated kinases modules associated with extracellular regulated kinases (ERK1/2) and Jnk3. Subsequently a number of other kinases have been shown to undergo β-arrestin-dependent regulation, including Akt, phosphatidylinositol-3kinase (PI3K), Lim-domain-containing kinase (LIMK), calcium calmodulin kinase II (CAMKII), and calcium calmodulin kinase kinase β (CAMKKβ). Some are positively and some negatively regulated by β-arrestin association. One of the missing links to understanding these pathways is the molecular mechanisms by which the activity of these kinases is regulated. Do β-arrestins merely serve as scaffolds to bring enzyme and substrate together or do they have a direct effect on the enzymatic activities of target kinases? Recent evidence suggests that both mechanisms are involved and that the mechanisms by which β-arrestins regulate kinase activity varies with the target kinase. This review discusses recent advances in the field focusing on 5 kinases for which considerable mechanistic detail and specific sites of interaction have been elucidated.

Calcium-Calmodulin Competes with Actin for Binding to the M-Domain of Cardiac Myosin Binding Protein-C

The regulatory M-domain of cardiac myosin binding protein-C (cMyBP-C) binds to myosin, actin, and to calmodulin when calcium is present (i.e., calcium-calmodulin), but it is unclear whether binding of all three ligands is independent or if binding interactions are competitive. Here we investigated whether calcium-calmodulin (Ca-Cam) binding to the M-domain affected the ability of the M-domain to bind to actin using cosedimentation binding and calmodulin-sepaharose pull-down assays. Results of actin cosedimentation binding assays showed that Ca-Cam significantly reduced specific binding of a recombinant protein containing three N-terminal domains of cMyBP-C (i.e., C1-M-C2, referred to as C1C2) when 10 μM calmodulin was present in the presence of calcium (pCa 3.0). In the absence of calcium (at pCa 10.0) calmodulin had no effect on C1C2 binding to actin. Increasing Ca-Cam concentrations to achieve higher molar ratios with respect to C1C2 further reduced the amount of C1C2 that bound to actin. Conversely, in calmodulin-sepharose pull-down experiments, binding of C1C2 to calmodulin was only modestly reduced in the presence of increasing concentrations of F-actin. Taken together, these results indicate that binding of Ca-Cam can compete with actin for binding to the M-domain. These results suggest a potential mechanism whereby the functional effects of cMyBP-C binding to actin can be regulated by calcium. This work supported by NIH HL080367.

CAMKIIα Catalytic Domain Dimerization is Regulated through T-Site Interactions

The calcium-calmodulin (CaM) dependent protein Kinase-II (CaMKII) regulates various cellular process and has been implicated in illness including schizophrenia, heart failure, arthritis, and even certain types of cancers. CaMKII subunits assemble to form a large dodecameric holoenzyme. The structure of a kinase subunit is organized into 3 domains, a catalytic domain, a regulatory domain, and an oligomerization domain. Each N-terminal catalytic domain harbors the kinase catalytic site, its ATP binding site, as well as the so-called ‘T-site’, thought to interact with the T286 autophosphorylation site in the regulatory domain. The T-site is responsible for mediating the interaction of the holoenzyme with other proteins, such as NMDA receptors, in response to activation. The catalytic domain is attached to an autoinhibitory regulatory domain that contains the T286 autophosphorylation site, and a CaM-binding site. In turn: the regulatory domain is itself tethered to a C-terminal oligomerization domain that organizes the enzyme into ring-shaped oligomers. We have previously shown in living cells using FRET analysis that catalytic domains form pairs in the intact auto-inhibited holoenzyme. In Hippocampal neurons, these pairs separate in response to calcium influx through NMDA-receptors, but it is not known if this is a direct response to CaM binding, or a down stream affect of kinase activation. Here using fluorescence polarization and fluctuation analysis (FPFA), a hybrid method that simultaneously measures homo-FRET and fluorescence correlation spectroscopy (FCS), we show that CaMKIINtide, a T-site ligand, disrupts catalytic domain pairing in the intact holoenzyme. This reaction is dose dependent but was only observed upon co-incubation with CaM to activate the enzyme. The holoenzyme can be activated without catalytic domain pair separation. We also show that T-site specific mutations at I205, stabilize catalytic domain pairing and counteract CaMKIINtide's ability to disrupt catalytic domain pairing.

Reducing the Number of Subunits in the CAMKIIα Holoenzyme Alters Catalytic Domain Pairing, Diffusion Time, and T286 Autophosphorylation

Calcium/Calmodulin (CaM) dependent protein kinase (CaMKII) is an important regulator of learning and memory, and it is thought to play a key role in sensing neuronal calcium oscillations. In cells, CaMKIIα primarily forms dodecamers comprised of two stacked rings of 6 subunits, each of which has 3 domains: an N-terminal catalytic domain, a C-terminal oligomerization domain, and between them, a regulatory domain. This regulatory domain activates the enzyme upon binding CaM and harbors a T286 autophosphorylation site that is associated with calcium independent persistent activation. To form the holoenzyme, the oligomerization domain of each subunit interacts with three other oligomerization domains: two laterally to form a ring of association domains, and one transversely to stabilize the double ring structure. Projecting outwards from the rings are catalytic domains, which form pairs, as indicated by homo-FRET analysis. Assemblies comprised of 8-14 subunits are also thought to exist, but it is not known if the number of subunits in the holoenzyme alters the activity or behavior of the catalytic domain pairs. To study the impact of subunit stoichiometry on catalytic domain pairing and T286 autophosphorylation, we generated mutants by altering oligomerization domain residues at the lateral interaction sites. Using fluorescence polarization and fluctuation analysis (FPFA) we simultaneously monitored homo-FRET between Venus-tagged catalytic domains, lateral diffusion of the holoenzyme, and the number of subunits present in these altered assemblies. We find that when the holoenzyme has fewer than 8 subunits, the holoenzyme diffusion time changes and homo-FRET between catalytic domains begins to attenuate. Finally, T286 autophosphorylation in a dimeric mutant holoenzyme is approximately half of that observed in the native holoenzyme. The structural implication of these changes will be discussed.

Physiological control of bioluminescence in a deep-sea planktonic worm, Tomopteris helgolandica Greeff, 1879

Tomopteris helgolandica Greeff 1879 (Tomopteridae) is a transparent holoplanktonic polychaete that can emit a bright light. In this study, we investigated the emission pattern and control of this deep-sea worm's luminescence. Potassium chloride depolarisation applied on anaesthetised specimens triggered a maximal yellow light emission from specific parapodial sites, suggesting that a nervous control pathway was involved. Pharmacological screening revealed a sensitivity to carbachol, which was confirmed by a dose-light response associated with a change in the light emission pattern, where physiological carbachol concentrations induced flashes and higher concentrations induced glows. The light response induced by its hydrolysable agonist, acetylcholine, was significantly weaker but was facilitated by eserine pretreatment. In addition, a specific inhibitory effect of tubocurarine was observed on carbachol-induced emission. Lastly, KCl- and carbachol-induced light responses were significantly reduced when preparations were pre-incubated in Ca(2+)-free artificial seawater or in different calcium channel blockers (verapamil, diltiazem) and calmodulin inhibitor (trifluoperazine) solutions. All of these results strongly suggest that T. helgolandica produces its light flashes via activation of nicotinic cholinergic receptors and a calcium-dependent intracellular mechanism involving L-type calcium channels.

Pro-apoptotic effect of anti-β1-adrenergic receptor antibodies in periodontitis patients

An anti-β1-adrenergic antibody from the sera of periodontitis patients (anti-β1-AR IgG) against the second extracellular loop of the human β1-adrenoceptor (β1-AR) has been shown to cause rat atria apoptosis. The anti-β1-AR IgG binds and activates atria β1-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The β1-AR and the post-receptor activation of calcium/calmodulin (CaM) lead to increased inducible nitric oxide synthase (iNOS) activity, with an increase in cyclic GMP (cGMP) accumulation as well as increased JNK phosphorylation and cyclic AMP (cAMP) production. We also observed an apoptotic effect of anti-β1-AR IgG, with increased generation of PGE2. Comparatively, xamoterol, an authentic β1-AR agonist, mimicked the autoantibody effect on rat atria β1-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria β1-AR, provoking apoptosis. This implicates a series of modulatory cardiac signaling events that could alter normal heart function and may occur with chronic stimulation of the atria β1-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.

Abstract 616: Caveolin-1 (CAV1) inhibits G Protein Coupled Receptor Kinase Type 4 (GRK4) kinase activity

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) has been shown to increase cAMP production and negatively regulate sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein-coupled receptor kinase type 4 (GRK4) which hyperphosphorylates and decreases D1R function, and the D1R uncoupling phenotype is fully reverted by reducing GRK4 expression with siRNA. We have also shown that CAV1 co-immunoprecipitates with GRK4, and that the normal D1R inhibition of the sodium transporter, NaKATPase alpha, can be blocked using siRNA to CAV1. We therefore hypothesized that CAV1 directly physically interacts with GRK4 and inhibits its kinase activity. Using purified human GRK4 and CAV1 proteins and an in-vitro peptide homogeneous kinase assay, we investigated the effects of caveolin-1 (CAV1) on the kinase activity of GRK4. By overexpressing and isolating a tandem affinity purified protein (TAP tagged) functional version of human CAV1 isolated from a human renal proximal tubule cell line, we were able to ensure that all of the post-translational modifications of the protein were present. Utilizing a peptide Ser/Thr kinase activity assay, we demonstrated that with an excess of CAV1, the kinase activity of GRK4 is inhibited by 37.8±1.3% (n=4, p<0.05). Using this same assay, we showed that purified human calcium calmodulin 1 (CALM1), a known inhibitor of GRK4 when using a complete cell lysate, does not significantly decrease the kinase activity of purified GRK4 using a CALM1 concentration over 100 times the published Kd value. This suggests that there is a missing co-factor or factors required for CALM1 to inhibit GRK4. In summary, we report for the first time that CAV1 inhibits the kinase activity of GRK4, and that a known inhibitor of GRK4, CALM1, does not directly inhibit GRK4 kinase activity using purified proteins in an in-vitro kinase activity assay.

Calcium–calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×10⁶/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×10⁶/ml. The final pH values after incubation at 30×10⁶ and 10×10⁶ sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO₂. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

Chronic Caffeine Treatment Prevents Stress-Induced LTP Impairment: the Critical Role of Phosphorylated CaMKII and BDNF

Caffeine has been reported to enhance cognition in animal and humans. Additionally, caffeine alleviates cognitive impairment associated with a number of disorders including Alzheimer's disease. The lipophilic nature of caffeine allows for rapid absorption into the bloodstream where it freely crosses the blood-brain barrier. Caffeine promotes dendritic spine growth in cultured hippocampal neurons, which suggests a neuroprotective effect. We examined the effect of chronic caffeine treatment on stress-induced suppression of long-term potentiation (LTP) and impairment of molecules of its signaling cascade. Rats were subjected to daily stress using the psychosocial stress paradigm (intruder model), in vivo recordings from area CA1 of the hippocampus of adult rat, and immunoblot analysis of essential signaling molecules. Caffeine prevented stress-induced LTP impairment. Western blot analysis showed reduction of the basal levels of the phosphorylated calcium calmodulin kinase II (P-CAMKII), total CaMKII, and brain-derived neurotrophic factor (BDNF) in area CA1 of stressed rats. These reductions were prevented by chronic caffeine treatment (0.33mg/L in drinking water). In addition, caffeine prevented the upregulation of calcineurin levels in stressed rats. High-frequency stimulation (HFS) normally increased P-CaMKII, total CaMKII, and calcineurin levels in control as well as in caffeine-treated stressed rats. However, in stressed rats, the same HFS induced increases in the levels of total CaMKII and calcineurin, but not those of P-CaMKII. The levels of signaling molecules may not reflect activities of these molecules. It appears that the neuroprotective effect of caffeine involves preservation of the levels of essential kinases and phosphatases in stressed rats. This may include preservation of basal levels of BDNF by chronic caffeine treatment in stressed animals. These findings highlight the critical role of P-CaMKII and BDNF in caffeine-induced prevention of stress-induced LTP impairment.

Heparin‐integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium–calmodulin dissociated actin from the MBP–lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

Neuronal NF-κB ablation does not influence neuro-axonal degeneration in experimental autoimmune demyelination

Neuro-axonal damage is a major hallmark of multiple sclerosis (MS). To date, not much is known on the underlying mechanisms of neuronal degeneration. In disease model myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE), there is a significant loss of alpha motorneurons in the cervical as well as thoracic and lumbar spinal cord. We further investigate the role of activated NF-κB for neuronal damage in a conditional ablation mouse model. A calcium calmodulin kinase II promoter-driven tetracycline transactivator is employed to regulate the expression of a human transdominant negative IκB-alpha mutant in the basal forebrain and selected neuronal subpopulations in the cerebellum and spinal cord including cerebellar Purkinje cells and spinal cord alpha motorneurons. In these mice with conditional neuronal NF-κB ablation, the clinical course of MOG-EAE, parameters of inflammation and axonal densities in the spinal cord white and grey matter as well as numbers of alpha motorneurons are not different to littermate controls. In conclusion, neuronal NF-κB ablation does not modulate neurodegeneration in autoimmune demyelination.

Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival

Transducin-like enhancer of split-1 (TLE1) plays a critical role in the regulation of neurogenesis by inhibiting the differentiation of neural progenitor cells into neurons. Although TLE1 is also expressed highly in the postnatal brain and through adulthood, its role in postmitotic neurons is not clear. Using cultures of cerebellar granule neurons, we show that expression of TLE1 is reduced in neurons primed to die. Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas suppression of TLE1 expression in otherwise healthy neurons induces cell death. These results show that TLE1 is necessary for the maintenance of neuronal survival. Experiments using pharmacological inhibitors as well as expression of point mutants indicate that phosphorylation of TLE1 by casein kinase-2 (CK2) at Ser-239 and Ser-253 is necessary for its survival-promoting activity. TLE1-mediated survival is also inhibited by pharmacological inhibition of PI3K-Akt signaling but not by inhibitors of Raf-MEK-ERK signaling or other molecules, including histone deacetylases, calcium calmodulin kinase, or CK1. The survival-promoting activity of TLE1 depends critically on interaction with FoxG1, another protein involved in the regulation of neurogenesis and shown previously to promote survival of postmitotic neurons. Likewise, the ability of FoxG1 to promote neuronal survival depends on TLE1. Taken together, our study demonstrates that TLE1 cooperates with FoxG1 to promote neuronal survival in a CK2- and PI3K-Akt-dependent manner.

α-Calcium calmodulin kinase II modulates the temporal structure of hippocampal bursting patterns.

The alpha calcium calmodulin kinase II (α-CaMKII) is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with α-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (α-CaMKIIT305D mutants), that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that α-CaMKIIT305D mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of α-CaMKIIT305D mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in α-CaMKIIT305D mutants showed higher variability than controls. These results provide in vivo evidence that besides its well-known function in synaptic plasticity, α-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.

Calcium–calmodulin signaling induced by epithelial cell differentiation upregulates BRAK/CXCL14 expression via the binding of SP1 to the BRAK promoter region

The chemokine BRAK/CXCL14 (BRAK) is expressed in normal squamous epithelium, but is not expressed or is expressed at negligible levels in head and neck squamous cell carcinoma. Malignant cells are known to be dedifferentiated compared with normal epithelial cells, suggesting a role for differentiation cues in the expression of BRAK. Thus, we examined the relationship between BRAK expression and stages of differentiation level in epithelial cells. Immunohistochemical analysis showed that BRAK protein was expressed in cells above the spinous cell layer in normal epithelia. In HSC-3 cells in culture, expression of BRAK mRNA was significantly upregulated by cell contact in a cell density-dependent manner, and mRNA expression of cell differentiation markers such as involucrin, cystatin-A, TGM1, TGM3, and TGM5 was concomitantly augmented. Furthermore, the upregulation of BRAK induced by cell contact was suppressed by chlorpromazine, a specific inhibitor of calmodulin. We previously reported that GC boxes and a TATA-like sequence in the BRAK promoter region are associated with the expression of BRAK. Using a promoter assay and ChIP, we demonstrated that binding of the stimulating protein-1 (SP1) transcription factor to a GC box upstream of the BRAK transcription start site was necessary for cell density-dependent upregulation of BRAK. These results indicated that upregulation of BRAK was accompanied by differentiation of epithelial cells induced by calcium/calmodulin signaling, and that SP1 binding to the BRAK promoter region played an important role in this signaling.

Involvement of calcium and calmodulin signaling in adaptation to heat stress-induced oxidative stress in Solanum lycopersicum L. leaves

Heat stress is a common form of stress suffered by plants. Therefore, plants have evolved mechanisms to cope with the problems caused by high temperatures. In this study, the involvement of calcium ion and calmodulin (Ca 2+ -CaM) in the protection against heat stress-induced oxidative damage in tomato ( Solanum lycopersicum L.) seedlings was investigated. Treatment of tomato leaves with short-term heat stress led to significant increase in the level of malondialdehyde (MDA) and protein oxidation (in terms of carbonyl groups), two important parameters of oxidative stress. However, such oxidative processes were mitigated by pretreatment with Ca 2+ and aggravated by pretreatment with the Ca 2+ chelator ethylene glycol-bis(β-amino ethyl ether)-N,N,N’,N’-tetraacetid acid (EGTA), the Ca 2+ channel blockers lanthanum chloride (LaCl 3 ), or the CaM antagonists chlorpromazine (CPZ), trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesul-fonamide (W7). Besides, Ca 2+ pretreatment significantly reduced the accumulation of reactive oxygen species (ROS), O 2 .- and H 2 O 2 and remarkably induced the total activities of the antioxidant enzymes superoxide dismutase (SOD) and ascorbate peroxidase (APX) under heat stress, while EGTA, LaCl 3 , CPZ, TFP and W7 pretreatment reversed them. The results obtained here suggest that Ca 2+ -CaM signaling pathways are involved in resistance to heat stress-induced oxidative stress by regulation of antioxidant mechanisms in tomato leaves. Key words: Calcium, calmodulin, heat stress, oxidative stress, Solanum lycopersicum L.

Spatiotemporal maps of CaMKII in dendritic spines

The calcium calmodulin dependent kinase (CaMKII) is important for long-term potentiation at dendritic spines. Photo-activatable GFP (PaGFP) - CaMKII fusions were used to map CaMKII movements between and within spines in dissociated hippocampal neurons. Photo-activated PaGFP (GFP*) generated in the shaft spread uniformly, but was retained for about 1s in spines. The differential localization of GFP*-CaMKII isoforms was visualized with hundred nanometer precision frame to frame using de-noising algorithms. GFP*-CaMKIIα localized to the tips of mushroom spines. The spatiotemporal profiles of native and kinase defective GFP*-CaMKIIβ, differed markedly from GFP*-CaMKIIα and mutant GFP*-CaMKIIβ lacking the association domain. CaMKIIβ bound to cortical actin in the dendrite and the stable actin network in spine bodies. Glutamate produced a transiently localized GFP*-CaMKIIα fraction and a soluble GFP*-CaMKIIβ fraction in spine bodies. Single molecule simulations of the interplay between diffusion and biochemistry of GFP* species were guided by the spatiotemporal maps and set limits on binding parameters. They highlighted the role of spine morphology in modulating bound CaMKII lifetimes. The long residence times of GFP*-CaMKIIβ relative to GFP*-CaMKIIα followed as consequence of more binding sites on the actin cytoskeleton than the post-synaptic density. These factors combined to retain CaMKII for tens of seconds, sufficient to outlast the calcium transients triggered by glutamate, without invoking complex biochemistry.