Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.     

Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice

Parkinson's disease (PD) patients frequently reveal deficit in cognitive functions during the early stage in PD. The dopaminergic neurotoxin, MPTP-induced neurodegeneration causes an injury of the basal ganglia and is associated with PD-like behaviors. In this study, we demonstrated that deficits in cognitive functions in MPTP-treated mice were associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and impaired long-term potentiation (LTP) induction in the hippocampal CA1 region. Mice were injected once a day for 5days with MPTP (25mg/kg i.p.). The impaired motor coordination was observed 1 or 2week after MPTP treatment as assessed by rota-rod and beam-walking tasks. In immunoblotting analyses, the levels of tyrosine hydroxylase protein and CaMKII autophosphorylation in the striatum were significantly decreased 1week after MPTP treatment. By contrast, deficits of cognitive functions were observed 3-4weeks after MPTP treatment as assessed by novel object recognition and passive avoidance tasks but not Y-maze task. Impaired LTP in the hippocampal CA1 region was also observed in MPTP-treated mice. Concomitant with impaired LTP induction, CaMKII autophosphorylation was significantly decreased 3weeks after MPTP treatment in the hippocampal CA1 region. Finally, the reduced CaMKII autophosphorylation was closely associated with reduced AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) phosphorylation in the hippocampal CA1 region of MPTP-treated mice. Taken together, decreased CaMKII activity with concomitant impaired LTP induction in the hippocampus likely account for the learning disability observed in MPTP-treated mice.     

Dysregulated phosphorylation of Ca(2+) /calmodulin-dependent protein kinase II-α in the hippocampus of subjects with mild cognitive impairment and Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder and the most prevalent senile dementia. The early symptom of memory dysfunction involves synaptic loss, thought to be mediated by soluble amyloid-beta (Aβ) oligomers. These aggregate species target excitatory synapses and their levels correlate with disease severity. Studies in cell culture and rodents have shown that oligomers increase intracellular calcium (Ca(2+)), impairing synaptic plasticity. Yet, the molecular mechanism mediating Aβ oligomers' toxicity in the aged brain remains unclear. Here, we apply quantitative immunofluorescence in human brain tissue from clinically diagnosed mild cognitive impaired (MCI) and AD patients to investigate the distribution of phosphorylated (active) Ca(2+) /calmodulin-dependent protein kinase-α (p(Thr286)CaMKII), a critical enzyme for activity-dependent synaptic remodeling associated with cognitive function. We show that p(Thr286)CaMKII immunoreactivity is redistributed from dendritic arborizations to neural perikarya of both MCI and AD hippocampi. This finding correlates with cognitive assessment scores, suggesting that it may be a molecular read-out of the functional deficits in early AD. Treatment with oligomeric Aβ replicated the observed phenotype in mice and resulted in a loss of p(Thr286)CaMKII from synaptic spines of primary hippocampal neurons. Both outcomes were prevented by inhibiting the phosphatase calcineurin (CaN). Collectively, our results support a model in which the synaptotoxicity of Aβ oligomers in human brain involves the CaN-dependent subcellular redistribution of p(Thr286)CaMKII. Therapies designed to normalize the homeostatic imbalance of neuronal phosphatases and downstream dephosphorylation of synaptic p(Thr286)CaMKII should be considered to prevent and treat early AD.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

The differential effects of single or repeated restraint stress on kainic acid-induced neuronal death in the hippocampal CA3 region: the role of glucocorticoid and various signal molecules

The effect of stress mediators following the stress period and addition time is a controversial issue until now. Thus, we aim to clarify the differential effects of single restraint stress (SS) or repeated restraint stress (RS) on kainic acid (KA)-induced neuronal death especially as addressing not only the role of glucocorticoid (Gc) and its receptor but also the signal pathway leading to cAMP response element binding protein phosphorylation (pCREB) and its functional role during stress. In the present study, we found that although RS did not show any difference on serum Gc level and hippocampal Gc receptor level compared to SS, SS exacerbated KA-induced neuronal death in hippocampal CA3 region, but RS did not. Moreover, pre-treatment with RU 38486 (Gc receptor antagonist) abolished the effect of SS on KA-induced neuronal death without an effect on KA toxicity itself. Furthermore, RS aggravates KA-induced neuronal death when CREB phosphorylation was deprived by KN-93 (calcium/calmodulin-dependent protein kinase II inhibitor). However, other signal molecules inhibitors such as PD98059 (MEK1/2 inhibitor) and SP600125 (p-p38 inhibitor) have no effect on KA-induced neuronal death after RS although these signal molecule were increased during SS or RS. These findings suggest that pCREB expression via calcium/calmodulin-dependent protein kinase II phosphorylation during RS comprise one of the balancers against Gc induced by stress.     

Distinct signalling pathways control Toxoplasma egress and host‐cell invasion

Calcium signalling coordinates motility, cell invasion, and egress by apicomplexan parasites, yet the key mediators that transduce these signals remain largely unknown. One underlying assumption is that invasion into and egress from the host cell depend on highly similar systems to initiate motility. Using a chemical‐genetic approach to specifically inhibit select calcium‐dependent kinases (CDPKs), we instead demonstrate that these pathways are controlled by different kinases: both TgCDPK1 and TgCDPK3 were required during ionophore‐induced egress, but only TgCDPK1 was required during invasion. Similarly, microneme secretion, which is necessary for motility during both invasion and egress, universally depended on TgCDPK1, but only exhibited TgCDPK3 dependence when triggered by certain stimuli. We also demonstrate that egress likely comes under a further level of control by cyclic GMP‐dependent protein kinase and that its activation can induce egress and partially compensate for the inhibition of TgCDPK3. These results demonstrate that separate signalling pathways are integrated to regulate motility in response to the different signals that promote invasion or egress during infection by Toxoplasma gondii.     

Inhibition of Endogenous Phosphatase in a Postsynaptic Density Fraction Allows Extensive Phosphorylation of the Major Postsynaptic Density Protein

Abstract: The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the a subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

Chronic high cytosolic calcium decreases AICAR-induced AMPK activity via calcium/calmodulin activated protein kinase II signaling cascade

Calcium is important for muscle contraction and controls many cellular processes. Although there is evidence that calcium-mediated signals regulate AMP-activated protein kinase (AMPK) activity, the molecular mechanisms by which calcium regulates AMPK are poorly understood. To compare the function of sustained vs. intermittent calcium oscillations on AMPK activity and define specific signals in this pathway, we administered mice with aminoimidazole-carboxamide-ribonucleotide (AICAR) and caffeine with or without dantrolene. AMPK activity was increased by 10 d AICAR treatment (P < 0.01). Ten day caffeine treatment decreased AICAR-induced AMPK activity to control level. This repressed AMPK activity was blocked by dantrolene. Different calcium frequencies were simulated in C2C12 myotubes by alternating media containing caffeine and dantrolene. Intermittent calcium oscillation increased AMPK activity compared to control (P < 0.05), whereas sustained calcium oscillation decreases AICAR-induced AMPK activity to control level. This result suggests a biphasic control of AMPK activity by calcium. Knockdown of CaMKII expression by short-hairpin RNA resulted in increased AMPK phosphorylation by AICAR even in the presence of caffeine. These data show different calcium oscillations elicit distinct responses in muscle cells suggesting that the negative effects of chronic calcium treatment on AMPK activity is partly mediated through the CaMKII signals.