Calmodulin-dependent Protein Kinase Type II Research Articles

Electroacupuncture at GB20 improves cognitive ability and synaptic plasticity via the CaM-CaMKII-CREB signaling pathway following cerebral ischemia-reperfusion injury in rats.

This study aimed to investigate the effects of electroacupuncture (EA) on cognitive recovery and synaptic remodeling in a rat model of middle cerebral artery occlusion (MCAO) followed by reperfusion and explore the possible mechanism. Focal cerebral ischemia was modeled in healthy adult Sprague-Dawley rats by MCAO. The MCAO rats were classified into four groups: sham, MCAO, MCAO + GB20 (receiving EA at GB20) and MCAO + NA (receiving EA at a "non-acupoint" location not corresponding to any traditional acupuncture point location about 10 mm above the iliac crest). Neurological deficit scores and behavior were assessed before and during the treatment. After intervention for 7 days, the hippocampus was dissected to analyze growth-associated protein (GAP)-43, synaptophysin (SYN) and postsynaptic density protein (PSD)-95 expression levels by Western blotting. Bioinformatic analysis and primary hippocampal neurons with calcium-voltage gated channel subunit alpha 1B (CACNA1B) gene overexpression were used to screen the target genes for EA against MCAO. Significant amelioration of neurological deficits and learning/memory were found in MCAO + GB20 rats compared with MCAO or MCAO + NA rats. Protein levels of GAP-43, SYN and PSD-95 were significantly improved in MCAO + GB20-treated rats together with an increase in the number of synapses in the hippocampal CA1 region. CACNA1B appeared to be a target gene of EA in MCAO. There were increased mRNA levels of CACNA1B, calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and cyclic adenosine monophosphate response element binding (CREB) and increased phosphorylation of CaM, CaMKII and CREB in the hippocampal region in MCAO + GB20 versus MCAO and MCAO + NA groups. CACNA1B overexpression modulated expression of the CaM-CaMKII-CREB axis. EA treatment at GB20 may ameliorate the negative effects of MCAO on cognitive function in rats by enhancing synaptic plasticity. EA treatment at GB20 may exert this neuroprotective effect by regulating the CACNA1B-CaM-CaMKII-CREB axis.

Activation of the N-methyl-D-aspartate receptor and calcium/calmodulin-dependent protein kinase IIα signal in the rostral anterior cingulate cortex is involved in pain-related aversion in rats with peripheral nerve injury

The rostral anterior cingulate cortex (rACC) of rat brain is associated with pain-related emotions. However, the underlying molecular mechanism remains unclear. Here, we investigated the effects of the N-methyl-D-aspartate (NMDA) receptor and Ca2+/Calmodulin-dependent protein kinase type II (CaMKII)α signal on pain-related aversion in the rACC of a rat model of neuropathic pain (NP). Mechanical and thermal hyperalgesia were examined using von Frey and hot plate tests in a rat model of NP induced by spared nerve injury (SNI) of the unilateral sciatic nerve. Bilateral rACC pretreatment with the CaMKII inhibitor tat-CN21 (derived from the cell-penetrating tat sequence and CaM-KIIN amino acids 43–63) or tat-Ctrl (the tat sequence and the scrambled sequence of CN21) was performed on postoperative days 29–35 in Sham rats or rats with SNI. Spatial memory performance was tested using an eight-arm radial maze on postoperative days 34–35. Pain-related negative emotions (aversions) were evaluated using the place escape/avoidance paradigm on postoperative day 35 following the spatial memory performance test. The percentage of time spent in the light area was used to assess pain-related negative emotions (i.e., aversion). The expression levels of the NMDA receptor GluN2B subunit, CaMKIIα, and CaMKII-Threonine at position 286 (Thr286) phosphorylation in contralateral rACC specimens were detected by Western blot or real time PCR following the aversion test. Our data showed that pretreatment of the rACC with tat-CN21 increased determinate behavior but did not alter hyperalgesia or spatial memory performance in rats with SNI. In addition, tat-CN21 reversed the enhanced CaMKII-Thr286 phosphorylation and had no effect on the upregulated expression of GluN2B, CaMKIIα protein, and mRNA. Our data suggested that activation of the NMDA receptor-CaMKIIα signal in rACC is associated with pain-related aversion in rats with NP. These data may provide a new approach for the development of drugs that modulate cognitive and emotional pain aspects.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Type II in Mediating Function and Dysfunction at Glutamatergic Synapses.

Glutamatergic synapses harbor abundant amounts of the multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Both in the postsynaptic density as well as in the cytosolic compartment of postsynaptic terminals, CaMKII plays major roles. In addition to its Ca2+-stimulated kinase activity, it can also bind to a variety of membrane proteins at the synapse and thus exert spatially restricted activity. The abundance of CaMKII in glutamatergic synapse is akin to scaffolding proteins although its prominent function still appears to be that of a kinase. The multimeric structure of CaMKII also confers several functional capabilities on the enzyme. The versatility of the enzyme has prompted hypotheses proposing several roles for the enzyme such as Ca2+ signal transduction, memory molecule function and scaffolding. The article will review the multiple roles played by CaMKII in glutamatergic synapses and how they are affected in disease conditions.

Abstract 11558: The Role of Oxidized CaMKII in Early Ischemia-Reperfusion Triggered Ventricular Arrhythmias

Introduction: Arrhythmias can complicate revascularization procedures after transmural infarctions and induce hemodynamic instability, but most cases can be adequately treated when they occur in hospital. However, arrhythmias that occur outside of hospital are potentially fatal. Arrhythmias in the early phase of reperfusion are caused by Ca 2+ overload and spontaneous diastolic release of Ca 2+ from the sarcoplasmic reticulum (SR). Indirect evidence suggests that reactive oxygen species (ROS) are important in this process by affecting the activity of key Ca 2+ handling proteins. Specifically, previous publications propose oxidation of the multifunctional Ca 2+ /calmodulin dependent protein kinase type II (CaMKII) to be a critical factor, and a potential therapeutic target. There is, however, a lack of data providing direct evidence for the importance of oxidized CaMKII (ox-CaMKII) for triggered arrhythmias in the early phase of reperfusion. Hypothesis: We hypothesized that ox-CaMKII increases diastolic SR Ca 2+ release events in the early phase of reperfusion. Methods: We evaluated the propensity for early reperfusion arrhythmias in mice resistant to oxidation of CaMKII in the heart (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT), by the use of Langendorff-perfused hearts exposed to ischemia-reperfusion, confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions, and immunoblotting. Results: Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in ventricular arrhythmias compared to WT during the early reperfusion phase, both at baseline and during beta-adrenoceptor stimulation with isoprenaline. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca 2+ -handling proteins. Ca 2+ imaging experiments without isoprenaline showed no significant difference in the incidence of spontaneous transients, Ca 2+ waves or Ca 2+ sparks between CaMKIIδ M281/282V and WT mice. Conclusions: Initial data indicate that ablation of CaMKII oxidation does not protect mouse hearts against arrhythmias in the early phase of reperfusion after ischemia.

The oxidation-resistant CaMKII-MM281/282VV mutation does not prevent arrhythmias in CPVT1.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased β‐adrenoceptor stimulation‐induced diastolic Ca2+ leak. We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca2+/calmodulin‐dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation‐resistant form of CaMKII protects mice carrying the CPVT1‐causative mutation RyR2‐R2474S (RyR2‐RS) against arrhythmias. Antioxidant treatment (N‐acetyl‐L‐cysteine) reduced the frequency of β‐adrenoceptor stimulation‐induced arrhythmogenic Ca2+ waves in isolated cardiomyocytes from RyR2‐RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation‐resistant CaMKII‐MM281/282VV variant (MMVV) were crossed with RyR2‐RS mice to create a double transgenic model (RyR2‐RS/MMVV). Wild‐type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2‐RS and RyR2‐RS/MMVV compared to wild‐type mice, both following a β‐adrenoceptor challenge (isoprenaline i.p.), and following treadmill exercise combined with a β‐adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2‐RS and RyR2‐RS/MMVV mice. Furthermore, no differences were observed in β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS/MMVV compared to RyR2‐RS. In conclusion, antioxidant treatment reduces β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS cardiomyocytes. However, oxidation‐resistant CaMKII‐MM281/282VV does not protect RyR2‐RS mice from β‐adrenoceptor stimulation‐induced Ca2+ waves or arrhythmias. Hence, alternative oxidation‐sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca2+ waves in cardiomyocytes from RyR2‐RS mice.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Altered Intracellular Calcium Homeostasis Underlying Enhanced Glutamatergic Transmission in Striatal-Enriched Tyrosine Phosphatase (STEP) Knockout Mice.

The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca2+ levels accompanied by an enhanced basal activity of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca2+ homeostasis and neurotransmitter release.

Ribosylation triggering Alzheimer's disease-like Tau hyperphosphorylation via activation of CaMKII.

Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer’s disease-like Tau hyperphosphorylation and diabetic encephalopathies.

Neuroprotective effect of muscone on glutamate-induced apoptosis in PC12 cells via antioxidant and Ca2+ antagonism

In the pathogenesis of cerebral ischemia, glutamate excitotoxicity activates N-methyl-d-aspartate (NMDA) receptors which induce calcium influx and oxidative stress. Muscone exerts potent neuroprotective activities on cerebral ischemia. However, its underlying mechanism is yet to be elucidated. In this study, we demonstrated that pretreatment with muscone in PC12 cells markedly ameliorated the loss of cell viability, mitochondrial membrane potential (MMP) collapse, the release of lactate dehydrogenase (LDH), Ca2+ overload, reactive oxygen species (ROS) generation, and cell apoptosis induced by glutamate. Furthermore, muscone also decreased NR1 (NMDA receptor subunit 1) protein expression, the ratio of Bax/Bcl-2 protein expression and prevented activitation of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and ASK1/JNK/p38 signaling pathways elicited by glutamate in PC12 cells. In conclusion, our results provided novel evidence that muscone protected PC12 cells against glutamate-induced apoptosis by attenuating ROS generation and Ca2+ influx, via NR1 and CaMKII-depended ASK-1/JNK/p38 signaling pathways.

Calcium dysregulation in atrial fibrillation: the role of CaMKII

Atrial fibrillation (AF) is the most frequently encountered clinical arrhythmia and is associated with increased morbidity and mortality. Ectopic activity and reentry are considered major arrhythmogenic mechanisms contributing to the initiation and maintenance of AF. In addition, AF is self-reinforcing through progressive electrical and structural remodeling which stabilize the arrhythmia and make it more difficult to treat. Recent research has suggested an important role for Ca2+-dysregulation in AF. Ca2+-handling abnormalities may promote ectopic activity, conduction abnormalities facilitating reentry, and AF-related remodeling. In this review article, we summarize the Ca2+-handling derangements occurring in AF and discuss their impact on fundamental arrhythmogenic mechanisms. We focus in particular on the role of the multifunctional Ca2+/calmodulin-dependent protein kinase type-II (CaMKII), which acts as a major link between Ca2+-dysregulation and arrhythmogenesis. CaMKII expression and activity are increased in AF and promote arrhythmogenesis through phosphorylation of various targets involved in cardiac electrophysiology and excitation-contraction coupling. We discuss the implications for potential novel therapeutic strategies for AF based on CaMKII and Ca2+-handling abnormalities.

Merger Fever: Can Two Separate Mechanisms Work Together to Explain Why We Drink?

Merger Fever: Can Two Separate Mechanisms Work Together to Explain Why We Drink?

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin‐dependent protein kinase II

Ca(2+) influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca(2+)/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein-alpha-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione-S-transferase pull-down assay. Thr286-autophosphorylated alpha-CaMKII or the autophosphorylation mimicking mutant, T286D-alpha-CaMKII, binds NR2B sequence independent of Ca(2+)/calmodulin unlike native wild-type alpha-CaMKII. We show enhancement of this binding by Ca(2+)/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca(2+)/calmodulin-independent binding whereas it allows the Ca(2+)/calmodulin-dependent binding of alpha-CaMKII in vitro. Similarly, the autonomously active mutants, T286D-alpha-CaMKII and F293E/N294D-alpha-CaMKII, exhibited Ca(2+)-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.

Flightless-I, a gelsolin family member and transcriptional regulator, preferentially binds directly to activated cytosolic CaMK-II

In order to evaluate links between Ca2+/calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T287D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T287D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of β-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences β-catenin dependent gene expression through Fli-I. Structured summaryMINT-6603828:delta CaMK-II (uniprotkb:Q6PHZ2) binds (MI:0407) to Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603847:delta CaMK-II (uniprotkb:Q6PHZ2) phosphorylates (MI:0217) MAP2 (uniprotkb:P20357) by protein kinase assay (MI:0424)MINT-6603768:beta CaMK-II (uniprotkb:P28652) physically interacts (MI:0218) with Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603759, MINT-6603776, MINT-6603786:delta CaMK-II (uniprotkb:Q6PHZ2) physically interacts (MI:0218) with Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603724:CaMK-II delta (uniprotkb:Q6PHZ2) physically interacts (MI:0218) with beta 5tubulin (uniprotkb:P99024), beta 2c tubulin (uniprotkb:P68372), alpha 1a tubulin (uniprotkb:P68369), b-Actin (uniprotkb:P60710), Tropomodulin-3 (uniprotkb:Q9JHJ0), Flightless-I (uniprotkb:Q9JJ28), FLAP1 (uniprotkb:Q3UZ39) and FLAP2 (uniprotkb:Q91WK0) by anti tag coimmunoprecipitation (MI:0007).

Calmodulin-dependent protein kinases phosphorylate gp130 at the serine-based dileucine internalization motif

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the low affinity LIF receptor (LIFR) and gp130. We previously demonstrated that LIF stimulation caused phosphorylation of gp130 at Ser782, adjacent to a dileucine internalization motif, and that transient expression of a mutant receptor lacking Ser782 resulted in increased cell surface expression and increased LIF-stimulated gene expression compared to wild-type receptor. Phosphorylation of Ser782 on gp130 fusion protein by LIF-stimulated 3T3-L1 cell extracts was inhibited 61% by autocamtide-2-related inhibitory peptide (AIP), a highly specific and highly effective inhibitor of calmodulin-dependent protein kinase type II (CaMKII). Purified rat forebrain CaMKII was also able to phosphorylate gp130 fusion protein at Ser782 in vitro. Furthermore, antibodies targeting CaMKII and CaMKIV were able to immunoprecipitate gp130 phosphorylating activity from LIF-stimulated 3T3-L1 lysates. While pretreatment of cells with the MAPKK inhibitors PD98059 and U0126 blocked phosphorylation of Ser782 prior to LIF stimulation, these inhibitors did not block Ser782 phosphorylation by LIF-stimulated 3T3-L1 cell extracts in vitro. These results show that CaMKII and possibly CaMKIV phosphorylate Ser782 in the serine-based dileucine internalization motif of gp130 via a MAPK-dependent pathway.

Involvement of ASK1 in Ca2+‐induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca2+ signalling regulates the ASK1-p38 MAP kinase cascade. Ca2+ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca2+/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca2+ signalling between CaMKII and p38 MAP kinase.

Axonal localization of delta Ca2+/calmodulin-dependent protein kinase II in developing P19 neurons

Ca2+/calmodulin-dependent protein kinase, type II (CaMK-II) is an enzyme encoded by four genes (α, β, γ and δ) and traditionally associated with synaptic function in the adult central nervous system, but also believed to play a role during neuronal development. P19 mouse embryonic cells are a model system for neurogenesis and primarily express isozymes of δ CaMK-II. It is not yet known whether or where δ CaMK-II is expressed in P19 neurons. Using an antibody specific for the δ CaMK-II C-terminal tail, we detected a 20-fold increase in levels of δ CaMK-II along axons after 8 days of development. This coincides with increased mRNA and protein levels of δC CaMK-II, which contains the alternative tail. This follows the initial stages of neurite outgrowth and β3 tubulin expression, which occur after 4 days. δ CaMK-II co-localizes with the axonal protein GAP-43, but not the dendritic microtubule-associated protein MAP-2, a known substrate of α CaMK-II. Like δ CaMK-II, GAP-43 shows increased expression after 8 days. These findings demonstrate developmental regulation of the alternative C-terminal δ CaMK-II exon and implicate endogenous δ CaMK-II in axonal development in embryonic cells.

?-Ca2+/calmodulin-dependent protein kinase II expression pattern in adult mouse heart and cardiogenic differentiation of embryonic stem cells

delta-isoforms of the Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) are considered to substantially influence cardiac functions. However, no data exist on the expression of these isoforms in the mouse heart. We analyzed the transcript pattern of non-neuronally expressed delta-isoforms in heart and skeletal muscle of adult mice by RT-PCR. For members of the delta-CaMKII subclass with both variable domains (subclass II), weak transcriptional expression of isoforms delta(2) and delta(3) was found in the heart. In skeletal muscle no delta(3)-specific transcript was detectable. In cardiac tissue, stronger signals result from amplifications of delta(9) and from members of the subclass I lacking the second variable domain. Western blotting was performed using a subclass II-specific antibody. In murine cardiac and skeletal muscle tissue a delta-CaMKII protein pattern was obtained similar to that described for rat. To gain insight into the expression of delta-CaMKII during the earliest steps of cardiogenic differentiation, we analyzed the transcript pattern of murine embryonic stem cell-derived cardiomyocytes in various differentiation stages. Reproducible RT-PCR signals could be obtained for delta(6) and delta(10), both belonging to the delta-CaMKII subclass I. Transcripts for delta(6) were ubiquitously expressed, whereas transcripts for delta(10) were detectable in increasing amounts after 7-10 days of the onset of cardiogenic differentiation. Our results point to a differentiation-dependent expression of the two delta-CaMKII subclasses, and also to differences in the expression of individual members of subclass I during the early stages of cardiogenic differentiation.

RA175, which is expressed during the neuronal differentiation of P19EC cells, temporally expressed during neurogenesis of mouse embryos

Ca2+/calmodulin-dependent protein kinase, type II (CaMK-II) is an enzyme encoded by four genes (α, β, γ and δ) and traditionally associated with synaptic function in the adult central nervous system, but also believed to play a role during neuronal development. P19 mouse embryonic cells are a model system for neurogenesis and primarily express isozymes of δ CaMK-II. It is not yet known whether or where δ CaMK-II is expressed in P19 neurons. Using an antibody specific for the δ CaMK-II C-terminal tail, we detected a 20-fold increase in levels of δ CaMK-II along axons after 8 days of development. This coincides with increased mRNA and protein levels of δC CaMK-II, which contains the alternative tail. This follows the initial stages of neurite outgrowth and β3 tubulin expression, which occur after 4 days. δ CaMK-II co-localizes with the axonal protein GAP-43, but not the dendritic microtubule-associated protein MAP-2, a known substrate of α CaMK-II. Like δ CaMK-II, GAP-43 shows increased expression after 8 days. These findings demonstrate developmental regulation of the alternative C-terminal δ CaMK-II exon and implicate endogenous δ CaMK-II in axonal development in embryonic cells.

Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression.

Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.

Calcium/Calmodulin-Dependent Protein Kinase Types II and IV Differentially Regulate CREB-Dependent Gene Expression

Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.