CaMKII Gene Research Articles

Specific CaMKIIs mediate convergent extension cell movements in early zebrafish development.

Noncanonical Wnts are morphogens that can elevate intracellular Ca2+, activate the Ca2+/calmodulin-dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation. Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1-3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant-negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes. Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE.

Daytime restriction of feeding prevents illuminated night-induced impairment of metabolism and sleep in diurnal zebra finches

We investigated whether nocturnal eating was causal to the impairment of metabolism and sleep disruption in diurnal animals exposed to illuminated nights. Adult zebra finches hatched and raised in 12 h light: 12 h darkness (LD) were exposed to 5-lux dim light at night (dLAN, two groups), with a control group maintained on LD. For the next 3 weeks, the food availability to one of the dLAN groups was restricted to the 12 h light period (dLAN -F); the other dLAN (dLAN +F) and LD groups were continued on ad lib feeding. In spite of similar food intakes, dLAN +F condition led to the fat accumulation and weight gain. These birds showed concurrent changes in hepatic expression of genes associated with carbohydrate and lipid metabolism, suggesting an enhanced gluconeogenesis and impaired fatty acids synthesis. Increased sirt1 mRNA levels indicated the activation of molecular mechanisms to counter-balance the metabolic damage under dLAN +F. Furthermore, reduced bout length and total duration of the nocturnal sleep suggested a poorer sleep in dLAN +F condition. Negative sleep effects of dLAN were supported by the lower hypothalamic expression of sleep promoting sik3 and camkii genes, and higher mRNA expression of awake promoting achm3 gene in dLAN +F, compared to the LD condition. Importantly, dLAN-induced negative effects in metabolism and sleep were alleviated in the dLAN –F group. These results suggest the role of timed feeding in alleviating the negative impact of illuminated nights in metabolism and sleep in diurnal zebra finches.

Pairing-Dependent Plasticity in a Dissected Fly Brain Is Input-Specific and Requires Synaptic CaMKII Enrichment and Nighttime Sleep.

In Drosophila, in vivo functional imaging studies revealed that associative memory formation is coupled to a cascade of neural plasticity events in distinct compartments of the mushroom body (MB). In-depth investigation of the circuit dynamics, however, will require an ex vivo model that faithfully mirrors these events to allow direct manipulations of circuit elements that are inaccessible in the intact fly. The current ex vivo models have been able to reproduce the fundamental plasticity of aversive short-term memory, a potentiation of the MB intrinsic neuron (Kenyon cells [KCs]) responses after artificial learning ex vivo. However, this potentiation showed different localization and encoding properties from those reported in vivo and failed to generate the previously reported suppression plasticity in the MB output neurons (MBONs). Here, we develop an ex vivo model using the female Drosophila brain that recapitulates behaviorally evoked plasticity in the KCs and MBONs. We demonstrate that this plasticity accurately localizes to the MB α′3 compartment and is encoded by a coincidence between KC activation and dopaminergic input. The formed plasticity is input-specific, requiring pairing of the conditioned stimulus and unconditioned stimulus pathways; hence, we name it pairing-dependent plasticity. Pairing-dependent plasticity formation requires an intact CaMKII gene and is blocked by previous-night sleep deprivation but is rescued by rebound sleep. In conclusion, we show that our ex vivo preparation recapitulates behavioral and imaging results from intact animals and can provide new insights into mechanisms of memory formation at the level of molecules, circuits, and brain state.SIGNIFICANCE STATEMENT The mammalian ex vivo LTP model enabled in-depth investigation of the hippocampal memory circuit. We develop a parallel model to study the Drosophila mushroom body (MB) memory circuit. Pairing activation of the conditioned stimulus and unconditioned stimulus pathways in dissected brains induces a potentiation pairing-dependent plasticity (PDP) in the axons of α′β′ Kenyon cells and a suppression PDP in the dendrites of their postsynaptic MB output neurons, localized in the MB α′3 compartment. This PDP is input-specific and requires the 3′ untranslated region of CaMKII. Interestingly, ex vivo PDP carries information about the animal's experience before dissection; brains from sleep-deprived animals fail to form PDP, whereas those from animals who recovered 2 h of their lost sleep form PDP.

CaMKII(δ) regulates osteoclastogenesis through ERK, JNK, and p38 MAPKs and CREB signalling pathway

Calcium/calmodulin-dependent protein kinases (CaMKs) are a group of important molecules mediating calcium signal transmission and have been proved to participate in osteoclastogenesis regulation. CaMKII, a subtype of CaMKs is expressed during osteoclast differentiation, but its role in osteoclastogenesis regulation remains controversial. In the present study, we identified that both mRNA and protein levels of CaMKII (δ) were upregulated in a time-dependent manner during osteoclast differentiation. CaMKII (δ) gene silencing significantly inhibited osteoclast formation, bone resorption, and expression of osteoclast-related genes, including nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and c-Src. Furthermore, CaMKII (δ) gene silencing downregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38, which were transiently activated by RANKL. Specific inhibitors of ERK, JNK, and p38 also markedly inhibited expression of osteoclast-related genes, osteoclast formation, and bone resorption like CaMKII (δ) gene silencing. Additionally, CaMKII (δ) gene silencing also suppressed RANKL-triggered CREB phosphorylation. Collectively, these data demonstrate the important role of CaMKII (δ) in osteoclastogenesis regulation through JNK, ERK, and p38 MAPKs and CREB pathway.

Genetic compensation of γ CaMKII, an evolutionarily conserved gene.

CaMKII is a Ca2+/CaM-dependent protein kinase encoded by a family of conserved genes found throughout all metazoan species and expressed from fertilization into adulthood. One of these genes, camk2g1, is particularly important during early development as determined by pharmacologic, dominant negative and antisense morpholino approaches in zebrafish. Four other teleost fish species (cavefish, medaka, stickleback, and tilapia), exhibit sequence conservation of camk2g1 and duplication of the same CaMKII genes. A homozygous mutant of camk2g1 was generated in zebrafish using TALEN technology but yielded none of the phenotypic alterations seen using all other approaches and was reproductively viable. However, these camk2g1 mutant embryos showed a 4-fold over-expression of its paralog camk2g2. None of the other camk2 genes showed such transcriptional elevation, in fact, some of these genes were suppressed to 10% of wild type levels. In contrast, G0 camk2g1 CRISPR/Cas9 embryos recapitulated nearly all of the altered phenotypes observed in camk2g1 morphants, including renal, aural and ciliary defects. These findings validate the importance of this gene family during early zebrafish development and provide evidence for gene-specific transcriptional cross-talk consistent with genetic compensation.

Longzhibu disease and its therapeutic effects by traditional Tibetan medicine: Ershi-wei Chenxiang pills.

Ershi-wei Chenxiang pills (ECP) or Aga Nixiu wan (ཨ་གར་ཉི་ཤུ།), composed of 20 Tibetan medicines, has the effect of promoting blood circulation to remove blood stasis. As a common and frequent prescription used by traditional Tibetan medicine in clinical treatment of Longzhibu disease (cerebral ischemia sequelae), it has a significant effect. However, its anti-cerebral ischemia mechanism is still unclear. The chemical components of ECP were determined by high-performance liquid chromatography and gas chromatography-mass spectrometry. SD rats were randomly divided into Sham, MCAO, Nim (20.00mg/kg), and ECP (1.33 and 2.00g/kg) groups, with 13 animals in each group. After 14 days of oral administration, we established a model of cerebral ischemia reperfusion injury by blocking the middle cerebral artery of rats. After 24h of reperfusion injury, we evaluated the protective effect of ECP on ischemic brain by neural function score, TTC, H&E and Nissl staining. TUNEL fluorescence, western blot and immunohistochemistry were used to detect the phenomenon of apoptosis and the expression of apoptosis-related proteins Bax, Bcl-2, Cyto-c and activated Caspase-3. Furthermore, western blot, qRT-PCR and immunohistochemistry were employed to detect CaMKⅡ, ATF4 and c-Jun gene and protein expression. ECP contains agarotetrol, eugenol, oleanolic acid, ursolic acid, dehydrodiisoeugenol, hydroxysafflor yellow A, kaempferide, gallic acid, alantolactone, isoalantolactone, costunolide, dehydrocostus lactone, brucine, strychnine, echinacoside, bilirubin and cholic acid. Compared with MCAO group, ECP can significantly ameliorate the neurological deficit of cerebral ischemia in rats and reduce the volume of cerebral infarction. Pathological and Nissl staining results showed that ECP sharply inhibited the inflammatory infiltration injury of neurons and increased the activity of neurons in comparation with the MCAO group. TUNEL fluorescence apoptosis results confirmed that ECP obviously inhibited the apoptosis of neurons. Meanwhile, the results of immunohistochemistry and western blot demonstrated that EPC can dramatically inhibit the expression of pro-apoptotic proteins Bax, Cyto-c and activated Caspase-3, while increase the level of anti-apoptotic protein Bcl-2. In addition, compared with MCAO group, CaMK Ⅱ gene and protein expression were improved significantly by ECP administration. while, the expression of ATF4 and c-Jun genes and proteins were decreased. In conclusion, this study preliminarily demonstrated that the protective effect of ECP on ischemic brain is related to the improvement of neurological deficit, reducing the size of cerebral infarction, improving the activity of neurons, inhibiting the mitochondrial apoptosis pathway by regulating the protein expression of CaMKⅡ, ATF4 and c-Jun. However, further in vivo and in vitro investigations are still needed to clarify the underlying mechanism of ECP in treating cerebral ischemia sequelae.

NMDA receptors on zebrafish Mauthner cells require CaMKII-α for normal development.

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

Toxicity of TDCPP and TCEP on PC12 cell: Changes in CAMKII, GAP43, tubulin and NF-H gene and protein levels

TDCPP and TCEP are two major types of organophosphorus flame retardants (OPFRs) that are bioaccumulative and persistent in the environment. The toxicity effects of TDCPP and TCEP on PC12 cell are not well understood. In the present study, we investigated morphology, viability and apoptosis in cultured PC12 cells in response to TDCPP and TCEP. The mRNA and protein expression levels of CAMKII, GAP43, tubulin and NF-H were quantified in PC12 cells treated with varying concentrations of the two agents. Results indicate that, upon treatment with the two OPFRs, cell growth decreased, apoptosis increased, morphology was altered and significant changes were found in the gene and protein levels. Treatment with TDCPP caused a reduction in the levels of each of the six proteins studied and in the gene levels of GAP43, NF-H and the two tubulins, but it resulted in an increase in CAMKII gene levels. Treatment with TCEP resulted in similar changes in gene levels to TDCPP and led to decreases in the protein levels of GAP43 and the tubulins while increasing the CAMKII and NF-H protein levels. These results suggest that changes in the gene and protein levels of the regulatory proteins (CAMKII, GAP43) and the structural proteins (tubulin, NF-H) are due to different mechanisms of the toxins, and these proteins may be useful biomarkers for the cytotoxicity and neurotoxicity.

Signal‐transduction pathways in hemolysis‐associated right‐ventricular failure (1097.12)

The pulmonary arterial pressure at which the right ventricle fails in patients with sickle‐cell disease occurs at markedly lower pressures than in pulmonary hypertension not associated with hemolysis. The mechanism by which the right ventricle fails and the reason that dysfunction occurs at relatively low pulmonary arterial pressures in sickle‐cell disease remains poorly understood. Preliminary data from our group suggests that hemolysis, through activation of Ras homolog gene family member A (RhoA), causes activation of NADPH oxidase 2 (Nox2) and calcium/calmodulin‐dependent protein kinase II in the right ventricle leading to the production of reactive oxygen species, downstream mitochondrial destruction and ultimately, maladaptive remodeling of the right ventricle. We proceeded to examine whether RhoA activation indeed activates CaMKII via Nox2 and to see if this signaling axis drives myocyte dysfunction as evidenced by eNOS uncoupling and mitochondrial dysfunction. Six hours following exposure of rat neonatal cardiac myocytes (RNCM) to lysed red cell plasma resulted in a marked increase in superoxide production (P<0.01, n=15) that was significantly reduced by co‐incubation with the Nox2ds peptide (P<0.001). Bioenergetic analyses of RNCM showed a significant decrease in oxygen consumption rate after exposure to hemolyzed blood. Transfection studies showed an increase in RhoA expression after silencing Nox2 and CaMKII genes, suggesting that if RhoA is linked to these proteins, its activation may be upstream of Nox2 and CaMKII. Ongoing studies using peptide inhibitors are being employed to further elucidate the roles of the aforementioned proteins in hemolysis‐associated myocyte dysfunction.

Abstract P183: Cardiac Deletion of CaMKII Delta and Gamma Protects Against Heart Failure Despite Activation of Calcineurin Signaling

CaMKII delta and gamma are the major CaMKII genes expressed in the heart, and both are up-regulated in response to pressure overload. Recently, we have demonstrated that CaMKII delta single knockout mice are protected against cardiac hypertrophy and remodeling. However, the role of CaMKII gamma and potential redundant functions of CaMKII delta and gamma are still elusive. The aim of the present study was to evaluate the function of CaMKII delta and gamma by a cardiomyocyte-specific double knockout mouse model(delta/gamma-CKO). Strikingly, whereas delta and gamma single knockout mice displayed only slightly reduced levels of cardiac phospholamban (PLN) phosphorylation at the CaMKII phosphorylation site threonin 17, in delta/gamma-CKO mice there was almost no residual PLN-threonin-17 phosphorylation detectable. Surprisingly and in contrast to delta and gamma single knockout mice, delta/gamma-CKO mice did develop cardiac hypertrophy after transverse aortic constriction (TAC). Despite cardiac hypertrophy we observed markedly reduced cardiac fibrosis and apoptosis. Microarray analysis revealed a distinct different gene expression profile pointing to an activation of calcineurin in delta/gamma-CKO mice after TAC. Phosphorylation of calcineurin at serine 197, which leads to an inactivation of its enzymatic activity, was almost abolished in delta/gamma-CKO mice. To test the therapeutical implications of a complete myocardial CaMKII knockout, an tamoxifen-inducible knockout system was established. Knockout of CaMKII delta and gamma was induced by administration of tamoxifen three weeks after TAC surgery. Whereas control mice did develop overt heart failure and cardiac remodeling 16 weeks after TAC, delta/gamma-iCKO mice recovered from cardiac dysfunction. Taken together, our mouse genetic studies demonstrate that CaMKII delta and gamma are promising drug targets to restore cardiac function after pathological stress. These data also unmask a cross talk of CaMKII to endogenous calcineurin signaling, which results in adaptive cardiac hypertrophy and not pathological remodeling.

Characterization and Evolution Analysis of CaMKII Gene in Pig and Other Species

Characterization and Evolution Analysis of CaMKII Gene in Pig and Other Species

Differential expression of CaMK‐II genes during early zebrafish embryogenesis

CaMK-II is a highly conserved Ca(2+)/calmodulin-dependent protein kinase expressed throughout the lifespan of all vertebrates. During early development, CaMK-II regulates cell cycle progression and "non-canonical" Wnt-dependent convergent extension. In the zebrafish, Danio rerio, CaMK-II activity rises within 2 hr after fertilization. At the time of somite formation, zygotic expression from six genes (camk2a1, camk2b1, camk2g1, camk2g2, camk2d1, camk2d2) results in a second phase of increased activity. Zebrafish CaMK-II genes are 92-95% identical to their human counterparts in the non-variable regions. During the first three days of development, alternative splicing yields at least 20 splice variants, many of which are unique. Whole-mount in situ hybridization reveals that camk2g1 comprises the majority of maternal expression. All six genes are expressed strongly in ventral regions at the 18-somite stage. Later, camk2a1 is expressed in anterior somites, heart, and then forebrain. Camk2b1 is expressed in somites, mid- and forebrain, gut, retina, and pectoral fins. Camk2g1 appears strongly along the midline and then in brain, gut, and pectoral fins. Camk2g2 is expressed early in the midbrain and trunk and exhibits the earliest retinal expression. Camk2d1 is elevated early at somite boundaries, then epidermal tissue, while camk2d2 is expressed in discrete anterior locations, steadily increasing along either side of the dorsal midline and then throughout the brain, including the retina. These findings reveal a complex pattern of CaMK-II gene expression consistent with pleiotropic roles during development.

Adhesion-dependent activation of CaMKII and regulation of ERK activation in vascular smooth muscle

Cell adhesion-dependent activation of ERK1/2 has been linked functionally to focal adhesion dynamics. We previously reported that in adherent vascular smooth muscle (VSM) cells, CaMKII mediates ERK1/2 activation in response to Ca(2+)-mobilizing stimuli. In the present study, we tested whether CaMKII regulates ERK1/2 signaling in response to VSM cell adhesion. Using an antibody that specifically recognizes CaMKII autophosphorylated on Thr(287), we determined that CaMKII is rapidly activated (within 1 min) after the adherence of cells on multiple ECM substrates. Activation of CaMKII on fibronectin was unaffected in cells overexpressing focal adhesion kinase (FAK)-related nonkinase (FRNK), an endogenous inhibitor of FAK. Furthermore, CaMKII was rapidly and robustly activated in VSM cells plated on poly-l-lysine. These results suggest that adhesion-dependent CaMKII activation is integrin independent. Adhesion-dependent FAK activation on fibronectin was not affected in cells treated with the selective CaMKII inhibitor KN-93 (30 muM) or in cells in which the expression of CaMKII with small interfering RNA (siRNA) was suppressed, although tyrosine phosphorylation of paxillin was inhibited in CaMKII-delta(2)-suppressed cells. Sustained ERK1/2 activation that was dependent on FAK activation (inhibited by FRNK) was also attenuated by CaMKII inhibition or siRNA-mediated gene silencing. Rapid ERK1/2 activation that preceded FAK and paxillin activation was detected upon VSM cell adhesion to poly-l-lysine, and this response was inhibited by CaMKII gene silencing. These results indicate that integrin-independent CaMKII activation is an early signal during VSM cell adhesion that positively modulates ERK1/2 signaling through FAK-dependent and FAK-independent mechanisms.

Post-natal developmental expression of αKAP splice variants in rabbit fast-twitch and slow-twitch skeletal muscle

AlphaKAP is a non-kinase product of a gene within alpha CaMKII gene, that anchors catalytic subunits to skeletal muscle sarcoplasmic reticulum. alphaKAP gene expresses two distinct mRNA transcripts by alternative splicing in rabbit fast muscle, rather than one, as an apparently fiber-type specific and species-specific property. We report on the expression profile of alphaKAP gene in fast and slow muscles of adult rats and in rabbit post-natal developing muscles, by RT-PCR analysis of muscle RNA, together with Western blot analysis of expressed alphaKAP protein isoforms. We show that alphaKAP isoform predominantly expressed in muscle of newborn rabbits, corresponds to the fast-adult isoform. In fast muscle, the transition from neonatal isoform to the common adult isoform takes place early after birth, and is never complete, unlike in slow muscle. Relevant to these findings, is the observation that neonatal/embryonic alphaKAP mRNA is highly expressed in rat myotubes grown in vitro. Together, our findings suggest that alphaKAP splicing pathways are primarily modulated according to muscle developmental stage.

Analysis of the functional maturation of olfactory neurons in chicks before and after birth.

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.

Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure.

Long-term deficits in cognitive function are the principal effects of lead (Pb2+) exposure in children and can be modeled in experimental animals. Current therapeutic approaches in the treatment of childhood Pb2+ intoxication are not effective in reversing learning deficits once they have occurred. We report that environmental enrichment reverses long-term deficits in spatial learning produced by developmental Pb2+ exposure in rats. Enhanced learning performance of Pb2+-exposed animals reared in an enriched environment was associated with recovery of deficits in N-methyl-D-aspartate receptor subunit 1 (NR1) mRNA and induction of brain-derived neurotrophic factor (BDNF) mRNA in the hippocampus. The effect of environmental enrichment on NR1 and BDNF gene expression was specific to Pb2+-exposed animals and was present in the absence of changes in the NR2B subunit of the N-methyl-D-aspartate receptor, GluR1, alpha CamKII, or PSD-95 gene expression measured in the same animals. Our findings demonstrate that the learning impairments and NR1 subunit mRNA deficits resulting from developmental Pb2+ exposure are reversible if the animals are provided with an enriched environment even after the exposure has occurred. We propose environmental enrichment as a basis for the treatment of childhood Pb2+ intoxication.

Dynamics of [formula omitted]/calmodulin-dependent protein kinase II following acute myocardial ischemia—translocation and autophosphorylation

Ca 2+/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca 2+ concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.

Dynamic Expression Pattern of Ca2+/Calmodulin-Dependent Protein Kinase II Gene in the Central Nervous System of Drosophila throughout Development

Calcium/calmodulin-dependent protein kinase II (CaM KII) is thought to be involved in the majority of the neuronal functions mediated by intracellular free Ca2+, and has been implicated in long-term potentiation, learning, and memory. In this work, we have examined in detail the RNA expression pattern for the Drosophila CaM KII gene by in situ hybridization, during embryonic, larval, pupal, and adult stages. Our results indicate that expression of CaM KII was homogeneous in early embryos, but that during development the gene transcription rapidly became restricted to neuroblasts and their progeny in the nervous system. This predominant expression in the nervous system is maintained during late embryogenesis and post-embryonic development. A signal compartmentalization appeared in the larval central nervous system, where the CaM KII expression became progressively concentrated in the anterior ganglia. In the adult brain, a specific expression was more abundant in a subset of neurons around the central brain, particularly the mushroom bodies and the central complex, structures that play an important role in learning and memory.

Identification of novel human tumor cell-specific CaMK-II variants

CaMK-II (the (type II) multifunctional Ca 2+/CaM-dependent protein kinase) has been implicated in diverse neuronal and non-neuronal functions, including cell growth control. CaMKII expression was evaluated in a variety of human tumor cell lines using RT-PCR (reverse transcriptase coupled polymerase chain reaction). PCR primers which flanked the CaMK-II variable domain were used so that all possible variants of the four mammalian CaMK-II genes ( α, β, γ and δ) could be identified. 8 distinct CaMK-II isozymes were identified from human mammary tumor and neuroblastoma cell cDNA, each of which represented a variant of β, γ or δ CaMK-II. They included 2 β isozymes ( β e, β e′), 4 γ isozymes ( γ B, γ C, γ G, γ H) and 2 δ isozymes ( δ C, δ E) This is the first report of human β and δ CaMK-II sequences. A panel of human cell types was then screened for these CaMK-II isozymes. As expected, cerebral cortex predominately expressed α, β and δ A CaMK-II. In contrast, tumor cells, including those of neuronal origin, expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. Tumor cells of diverse tissue origin uniformly lacked α CaMK-II and expressed 1–2 β isozymes, at least 3 γ isozymes and 1–2 δ isozymes. When compared to undifferentiated fibroblasts, β e, β e′, γ G and γ H were preferentially expressed in tumor cells. CaMK-II immunoblots also indicated that neuroblastoma and mammary tumor cells express isozymes of CaMK-II not present in their non-transformed cell or tissue counterpart. The identification of these new, potential tumor-specific CaMK-II variants supports previous indications that CaMK-II plays a role in growth control. In addition, these results provide insight into both splice variant switching and variable domain structural similarities among all CaMK-II isozymes.

An alternative, nonkinase product of the brain-specifically expressed Ca2+/calmodulin-dependent kinase II alpha isoform gene in skeletal muscle.

The gene for the alpha isoform of Ca2+/calmodulin-dependent kinase II (alpha CaMKII) codes for a multifunctional protein kinase that is found exclusively in the brain. Here we show that in skeletal muscle, an alternative nonkinase product, hereafter referred to as alpha KAP (alpha CaMKII association protein), is expressed from the same gene. alpha KAP consists of a C-terminal region that is identical to the association domain of alpha CaMKII, with the exception of 11 amino acids inserted in the variable region. The N-terminal sequence of alpha KAP is highly hydrophobic and not present in any known CaMKII protein. The catalytic and regulatory domains of alpha CaMKII are missing in alpha KAP. Analysis of the exon-intron structure revealed that the alpha KAP transcript is derived from the alpha CaMKII gene by alternative promoter usage and RNA splicing. The transcriptional start site of alpha KAP mRNA is located within an intron of the alpha CaMKII gene. Therefore, the relationship between alpha KAP and alpha CaMKII is that of a gene within a gene. Immunostaining using anti-alpha KAP antibodies suggests that alpha KAP is associated with sarcomeres of skeletal muscle fibers. On the basis of its primary structure and specific location, the possible function of alpha KAP as an anchoring protein for CaMKII is discussed.