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Calmodulin-dependent Protein Kinase II (calmodulin-dependent + protein_kinase_ii)
Selected AbstractsCalcium,calmodulin-dependent protein kinase II phosphorylation modulates PSD-95 binding to NMDA receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2006Fabrizio Gardoni Abstract At the postsynaptic membrane of excitatory synapses, NMDA-type receptors are bound to scaffolding and signalling proteins that regulate the strength of synaptic transmission. The cytosolic tails of the NR2A and NR2B subunits of NMDA receptor bind to calcium,calmodulin-dependent protein kinase II (CaMKII) and to members of the MAGUK family such as PSD-95. In particular, although NR2A and NR2B subunits are highly homologous, the sites of their interaction with CaMKII as well as the regulation of this binding differ. We identified PSD-95 phosphorylation as a molecular mechanism responsible for the dynamic regulation of the interaction of both PSD-95 and CaMKII with the NR2A subunit. CaMKII-dependent phosphorylation of PSD-95 occurs both in vitro, in GST-PSD-95 fusion proteins phosphorylated by purified active CaMKII, and in vivo, in transfected COS-7 as well as in cultured hippocampal neurons. We identified Ser73 as major phosphorylation site within the PDZ1 domain of PSD-95, as confirmed by point mutagenesis experiments and by using a phospho-specific antibody. PSD-95 Ser73 phosphorylation causes NR2A dissociation from PSD-95, while it does not interfere with NR2B binding to PSD-95. These results identify CaMKII-dependent phosphorylation of the PDZ1 domain of PSD-95 as a mechanism regulating the signalling transduction pathway downstream NMDA receptor. [source] N-methyl- d -aspartate enhancement of the glycine response in the rat sacral dorsal commissural neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2000Tian-. Abstract The effect of N-methyl- d -aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 ,m NMDA to SDCN neurons reversibly potentiated Gly-activated Cl, currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 ,m), blocked the NMDA-induced potentiation of IGly, whereas 50 ,m CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+ -free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+,calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways. [source] Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase IIJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Donald M. Kuhn Abstract Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. TPH was once thought to be a single-gene product but it is now known to exist in two isoforms. TPH1 is found in the periphery and pineal gland whereas TPH2 is expressed specifically in the CNS. Both TPH isoforms are known to be regulated by protein kinase-dependent phosphorylation and the sites of modification of TPH1 by protein kinase A have been identified. While TPH2 is activated by calcium, calmodulin-dependent protein kinase II (CaMKII), the sites at which this isoform is modified are not known. Treatment of wild-type TPH2 with CaMKII followed by mass spectrometry analysis revealed that the enzyme was activated and phosphorylated at a single site, serine-19. Mutagenesis of serine-19 to alanine did not alter the catalytic function of TPH2 but this mutant enzyme was neither activated nor phosphorylated by CaMKII. A phosphopeptide bracketing phosphoserine-19 in TPH2 was used as an antigen to generate polyclonal antibodies against phosphoserine-19. The antibodies are highly specific for phosphoserine-19 in TPH2. The antibodies do not react with wild-type TPH2 or TPH1 and they do not recognize phophoserine-58 or phosphoserine-260 in TPH1. These results establish that activation of TPH2 by CaMKII is mediated by phosphorylation of serine-19 within the regulatory domain of the enzyme. Production of a specific antibody against the CaMKII phosphorylation site in TPH2 represents a valuable tool to advance the study of the mechanisms regulating the function of this important enzyme. [source] Regulation and function of Ca2+,calmodulin-dependent protein kinase II of fast-twitch rat skeletal muscleTHE JOURNAL OF PHYSIOLOGY, Issue 3 2007Adam J. Rose The activation and function of Ca2+,calmodulin-dependent kinase II (CaMKII) in contracting rat skeletal muscle was examined. The increase in autonomous activity and phosphorylation at Thr287 of CaMKII of gastrocnemius muscle in response to contractions in situ was rapid and transient, peaking at 1,3 min, but reversed after 30 min of contractions. There was a positive correlation between CaMKII phosphorylation at Thr287 and autonomous CaMKII activity. In contrast to the rapid and transient increase in autonomous CaMKII activity, the phosphorylation of the putative CaMKII substrate trisk95/triadin was rapid and sustained during contractions. There were no changes in CaMKII activity and phosphorylation or trisk95 phosphorylation in the resting contralateral muscles during stimulation. When fast-twitch muscles were contracted ex vivo, CaMKII inhibition resulted in a greater magnitude of fatigue as well as blunted CaMKII and trisk95 phosphorylation, identifying trisk95 as a physiological CaMKII substrate. In summary, skeletal muscle CaMKII activation was rapid and sustained during exercise/contraction and is mediated by factors within the contracting muscle, probably through allosteric activation via Ca2+,CaM. CaMKII may signal through trisk95 to modulate Ca2+ release in fast-twitch rat skeletal muscle during exercise/contraction. [source] Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl, channel geneTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Jorge Arreola Salivary gland acinar cells shrink when Cl, currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl, channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl, channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl, currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca2+ -calmodulin-dependent protein kinase II; however, the magnitude of the Ca2+ -dependent Cl, current was unchanged in the Clcn3,/- animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3,/- mice. Our results demonstrate that the ClC-3 Cl, channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva. [source] | ||||||||||