Calcium/calmodulin-dependent protein kinase II and synaptic plasticity

A prominent role for calcium/calmodulin-dependent protein kinase II (CaMKII) in regulation of excitatory synaptic transmission was proposed two decades ago when it was identified as a major postsynaptic density protein. Since then, fascinating mechanisms optimized to fine-tune the magnitude and locations of CaMKII activity have been revealed. The importance of CaMKII activity and autophosphorylation to synaptic plasticity in vitro, and to a variety of learning and memory paradigms in vivo has been demonstrated. Recent progress brings us closer to understanding the regulation of dendritic CaMKII activity, localization, and expression, and its role in modulating synaptic transmission and cell morphology.     

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: Effect of axotomy

Although Ca²⁺/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIα and IIβ are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIβ antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIα, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIα immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIα is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIα as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIβ down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIα appears to support axonal regrowth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 796–810, 1997     

Calcium/calmodulin-dependent protein kinase II: role in learning and memory

Numerous studies over the past decade have established a role(s) for protein phosphorylation in modulation of synaptic efficiency. This article reviews this data and focuses on putative functions of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) which is highly concentrated at these synapses which utilize glutamate as the neurotransmitter. Evidence is presented that CaM-kinase II can phosphorylate these glutamate receptor/ion channels and enhance the ion current flowing through them. This may contribute to mechanisms of synaptic plasticity that are important in cellular paradigms of learning and memory such as long-term potentiation in the hippocampus.     

Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs

cDNA clones for a fifth polypeptide of rat brain calmodulin-dependent protein kinase II were isolated and sequenced. The cDNA sequence encoded a polypeptide, designated delta, consisting of 533 amino acid residues with a molecular weight of 60,080. Comparison of amino acid sequences of this and alpha, beta, beta', and gamma polypeptides of calmodulin-dependent protein kinase II reveals marked homology among them. The mRNAs for delta were expressed in rat brain tissues with different regional specificities. The distribution of alpha, beta/beta', gamma, and delta mRNAs in cerebrum, skeletal muscle, diaphragm, heart, small intestine, uterus, aorta, liver, kidney, lung, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.9-kilobase (kb) RNA species hybridizable with a probe for gamma was found in all the tissues examined, and 4.0-4.2-kb RNA species hybridizable with a probe for delta were found in all the tissues examined except for liver, while a 4.8-kb RNA species hybridizable with a probe for alpha and a 4.2-kb RNA species hybridizable with a probe for beta were present in brain but not in the other tissues. With the alpha probe, however, a 4.1- and 2.6-kb RNA species were both detected in skeletal muscle and diaphragm. With the beta probe, a 4.3-kb RNA in skeletal muscle and diaphragm, 2.9-kb RNA in small intestine, and 4.0-kb RNA in testis were detected. With the delta probe, a 3.5-kb RNA in heart and 1.8-kb RNA in testis were detected. Thus, gamma and delta mRNAs were expressed in various tissues, while alpha and beta/beta' mRNAs were primarily, if not exclusively, expressed in brain.     

Calcium/calmodulin-dependent protein kinase II phosphorylation drives synapse-associated protein 97 into spines

Synapse-associated protein 97 (SAP97) has been involved in the correct delivery and clustering of glutamate ionotropic receptors to the postsynaptic compartment. Here we demonstrate that synaptic trafficking of SAP97 itself was modulated by calcium/calmodulin-dependent protein kinase II (CaMKII) in cultured hippocampal neurons. CaMKII activation led to increased targeting of SAP97 into dendritic spines, whereas CaMKII inhibition was responsible for SAP97 high colocalization in the cell soma with the endoplasmic reticulum protein disulfide-isomerase. No effect was detected for other members of the membrane-associated guanylate kinase protein family, such as SAP102 and PSD-95. Transfection of activated alphaCaMKII T286D dramatically increased concentration of both endogenous and transfected SAP97 at postsynaptic terminals. In vitro CaMKII phosphorylation of the SAP97 N-terminal fusion protein and metabolic labeling of transfected COS7 cells indicated SAP97-Ser-39 as a CaMKII phosphosite in the SAP97 protein sequence. Moreover, transfection in hippocampal neurons of SAP97 mutants that blocked or mimicked Ser-39 phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. Further, CaMKII-dependent SAP97-Ser-39 phosphorylation determined a redistribution of the glutamate receptor subunit (GluR1) of the AMPA receptor. In conclusion, our data show that CaMKII-dependent SAP97-Ser-39 phosphorylation regulates the association of SAP97 with the postsynaptic complex, thus providing a fine molecular mechanism responsible for the synaptic delivery of SAP97 interacting proteins, i.e. ionotropic glutamate receptor subunits.     

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.     

Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel

Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been postulated to regulate the ether à go-go (eag) potassium channel in Drosophila. Inhibition of CaMKII and mutation of the eag gene both cause hyperexcitability at the larval neuromuscular junction (NMJ) and memory formation defects in the adult. In this study, we identify a single site, threonine 787, as the major CaMKII phosphorylation site in Eag. This site can be phosphorylated by CaMKII both in a heterologous cell system and in vivo at the larval NMJ. Expression of Eag in Xenopus oocytes was used to assess the function of phosphorylation. Injection of either a specific CaMKII inhibitor peptide or lavendustin C, another CaMKII inhibitor, reduced Eag current amplitude acutely. Mutation of threonine 787 to alanine also reduced amplitude. Moreover, both CaMKII inhibition and the alanine mutation accelerated inactivation. The reduction in current amplitudes and the accelerated inactivation of dephosphorylated Eag channels would result in decreased outward potassium currents and hyperexcitability at presynaptic terminals and, thus, are consistent with the NMJ phenotype observed when CaMKII is inhibited. These results show that Eag is a substrate of CaMKII and suggest that direct modulation of potassium channels may be an important function of this kinase.     

Calcium/calmodulin-dependent protein kinase kinase 2 regulates macrophage-mediated inflammatory responses

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously, we showed that loss of CaMKK2 protected mice from high-fat diet (HFD)-induced obesity and glucose intolerance. However, although pair feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose-tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytose bacteria, and synthesize cytokines in response to the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives.     

Calcium/calmodulin-dependent protein kinase I in Xenopus laevis

Calcium/calmodulin (CaM) dependent protein kinase I (CaM-KI) is a member of a well-defined multi-functional CaM-K family, but its physiological and developmental functions have yet to be determined. Here, we have cloned two cDNAs encoding CaM-KI from a Xenopus laevis (X. laevis) oocyte cDNA library. One is a novel isoform of CaM-KI, named CaM-KI LiKbeta (XCaM-KI LiKbeta). The other is an alpha isoform of CaM-KI (XCaM-KIalpha), which is a highly related to previously cloned mammalian isoform. XCaM-KIalpha was constantly expressed through embryogenesis, whereas XCaM-KI LiKbeta expression dramatically increased in the neurula stage. Both XCaM-KI isoforms exhibited kinase activity in a Ca(2+)/CaM-dependent manner. Overexpression of a constitutively active mutant of CaM-KI isoforms inhibited cell cleavage in X. laevis embryos and caused a marked change of cell morphology in Hela cells. Taken together, these results suggest that CaM-KI plays a role in cell-structure regulation during early embryonic development.     

Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation

Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1.     

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.     

Calcium . calmodulin-dependent protein kinase II and calcium . phospholipid-dependent protein kinase activities in rat tissues assayed with a synthetic peptide

Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases.     

Calcium/calmodulin-dependent protein kinase IV limits organ damage in hepatic ischemia-reperfusion injury through induction of autophagy

Sterile inflammatory insults, such as ischemia-reperfusion (I/R) injury, result from pathogenic factors, including damage-associated molecular pattern signaling, activation of innate immunity, and upregulation of proinflammatory cytokines. At the same time, a number of protective, or prosurvival, pathways are also activated, and the extent of end-organ damage is ultimately determined by the balance between these two systems. In liver I/R, members of the calcium/calmodulin-dependent protein kinase (CaMK) family are known to be activated, but their individual roles are largely unknown. In this study, we show that one CaMK member, CaMKIV, is protective in hepatic I/R by activating the prosurvival pathway of autophagy in hepatocytes. CaMKIV knockout mice experience significantly worse organ damage after I/R and are deficient in hepatocyte autophagic signaling. Restoration of autophagic signaling with rapamycin reduces organ damage in CaMKIV knockout mice to wild-type levels. In vitro, we show that CaMKIV activation induces autophagy in mouse hepatocytes, and that CaMKIV activation protects hepatocytes from oxidative stress-induced cell death. In conclusion, the protective autophagic signaling pathway serves to reduce organ damage following I/R and is regulated by activation of CaMKIV signaling in hepatocytes.     

Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells

Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.     

Investigation of neuronal cell type-specific gene expression of Ca2+/calmodulin-dependent protein kinase II

The promoter activity of the rat Ca²⁺/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5′-flanking region of α and β isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5′-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types. Published: April 12, 2002