CaM kinase II


Table of contents

CaM kinase and the cell cycle

Calmodulin (CaM) has been shown to be essential for cell cycle progression in eukaryotic cells, being required at the G1/S, G2/M, and metaphase-anaphase transitions. Little is known about the the specific CaM-dependent enzymes that mediate Ca2+/CaM signaling to effect cell proliferation. Inhibition of calmodulin kinase II (CaMKII) in HeLa cells using the CaMKII inhibitor KN-93 causes cell cycle arrest, demonstrating that CaMKII is required for cell cycle progression. Detailed analysis of arrest cells suggests that CaMKII is required for the initiation of DNA synthesis. Cells treated with KN-93 arrest with a G1 DNA content, but they also show elevated cyclin-dependent histone H1 kinase activity, suggesting that CaMKII may act at a point very close to the onset of DNA synthesis in mammalian cells (Rasmussmen. 1995).

The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 reversibly arrests mouse and human cells in the G1 phase of the cell cycle. The stimulation of Ca(2+)-independent (autonomous) CaMK-II enzymatic activity, a barometer of in situ activated CaMK-II, is prevented by the same KN-93 concentrations that cause G1 phase arrest. KN-93 causes the retinoblastoma protein pRB to become dephosphorylated and the activity of both cdk2 and cdk4, two potential pRb kinases, to decrease. Neither the activity of p42MAP kinase, an early response G1 signaling molecule, nor the phosphorylation status or DNA-binding capability of the transcription factors serum response factor and cAMP responsive element-binding protein is altered during this G1 arrest. The protein levels of cyclin-dependent kinase 2 (cdk2) and cdk4 are unaffected during this G1 arrest and the total cellular levels of the cdk inhibitors p21cip1 and p27kip1 are not increased. Instead, the cdk4 activity decreases resulting from KN-93 are the result of a 75% decrease in cyclin D1 levels. In contrast, cyclin A and E levels are relatively constant. Cdk2 activity decreases are primarily the result of enhanced p27kip1 association with cdk2/cyclin E. All of these phenomena are unaffected by KN-93's inactive analog, KN-92, and are reversible upon KN-93 washout. The kinetics of recovery from cell cycle arrest are similar to those reported for other G1 phase blockers. These results suggest a mechanism by which G1 Ca2+ signals can be linked via calmodulin-dependent phosphorylations to the cell cycle-controlling machinery through cyclins and cdk inhibitors (Morris, 1998).

Elevation of intracellular free calcium causes mouse egg activation by initiating a cascade of interacting signaling pathways that, in unison, act to remodel the cytoplasmic compartment and the nuclear compartment of the egg. Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is tightly associated with the meiotic spindle and 5 min after egg activation there is a transient, tight association of calmodulin (colocalized with CaM kinase II) on the meiotic spindle. These correlative observations led to testing whether activation of CaM kinase II mediates the chromosomal transit into an anaphase configuration. Calcium and calmodulin, at physiological levels, along with ATP are capable of driving the spindle (with its associated CaM kinase II) into an anaphase configuration in a permeabilized egg system. The transit into anaphase is dependent on the presence of both calcium and calmodulin and occurs normally when they are present at a ratio of 4 to 1. Peptide and pharmacologic inhibitors of CaM kinase II block the transit into anaphase, both in the permeabilized egg system and in living eggs (inhibitors of protein kinase C do not block the transit into anaphase). Using a biochemical approach it was confirmed that CaM kinase II increases in activity 5 min after egg activation and a second increase occurs 45 min after activation at the approximate time that the contractile ring of the second polar body is constricting. This corresponds to the approximate time when calmodulin and CaM kinase II colocalize at several points in the activated egg, including the region containing midzone microtubules. CaM kinase II appears localized on midzone microtubules as soon as they form and may have a role in specifying the position of the contractile ring of the second polar body (Johnson, 1999).

The intracellular levels of cAMP play a critical role in the meiotic arrest of mammalian oocytes. However, it is debated whether this second messenger is produced endogenously by the oocytes or is maintained at levels inhibitory to meiotic resumption via diffusion from somatic cells. Adenylyl cyclase genes and corresponding proteins are expressed in rodent oocytes. The mRNA coding for the AC3 isoform of adenylyl cyclase was detected in rat and mouse oocytes by RT-PCR and by in situ hybridization. The expression of AC3 protein was confirmed by immunocytochemistry and immunofluorescence analysis in oocytes in situ. Cyclic AMP accumulation in denuded oocytes is increased by incubation with forskolin, and this stimulation is abolished by increasing intraoocyte Ca2+ with the ionophore A23187. The Ca2+ effects are reversed by an inhibitor of Ca2+, calmodulin-dependent kinase II. These regulations of cAMP levels indicate that the major cyclase that produces cAMP in the rat oocyte has properties identical to those of recombinant or endogenous AC3 expressed in somatic cells. Furthermore, mouse oocytes deficient in AC3 show signs of a defect in meiotic arrest in vivo and accelerated spontaneous maturation in vitro. Collectively, these data provide evidence that an adenylyl cyclase is functional in rodent oocytes and that its activity is involved in the control of oocyte meiotic arrest (Horner, 2003).

Fertilization-induced intracellular calcium (Ca2+) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca2+ signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca2+ similar to those at fertilization stimulate oscillatory Ca2+/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca2+ signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca2+ levels and CaMKII activity in the same population of eggs demonstrates a rapid and transient enzyme response to each increase in Ca2+. Enzyme activity increased 370% during the first Ca2+ rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca2+ reached its peak value. Single fertilized eggs monitored for Ca2+ had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca2+ transients resulted in 39% and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca2+ and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca2+ in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation (Markoulaki, 2003).

The role of CaM kinase II in Wnt signaling

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium [Ca(2+)] and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs was tested. Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, the hypothesis that CamKII participates in axis formation in the early embryo was tested. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos reveals elevated activity on the prospective ventral side, which is suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants are consistent with this prediction, revealing that CamKII contributes to a ventral cell fate (Kuhl, 2000).

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Endogenous Wnt/ß-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. The data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/ß-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/ß-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/ß-catenin pathway upstream of ß-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/ß-catenin signaling cascade downstream of ß-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus (Kuhl, 2001).

Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor [beta(2)AR-Rfz-2] containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor [beta(2)AR] and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling (Ishitani, 2003; full text of article).
CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia

Intracellular Ca2+ signals influence gastrulation, neurogenesis and organogenesis through pathways that are still being defined. One potential Ca2+ mediator of many of these morphogenic processes is CaMK-II, a conserved calmodulin-dependent protein kinase. Prolonged Ca2+ stimulation converts CaMK-II into an activated state that, in the zebrafish, is detected in the forebrain, ear and kidney. Autosomal dominant polycystic kidney disease has been linked to mutations PKD2, a member of thhe Ca2+-permeable TRP superfamily of nonselective ion channels. Suppression of PKD2 in vertebrate model organisms results in kidney cysts. Both PKD2-deficient and CaMK-II-deficient zebrafish embryos fail to form pronephric ducts properly, and exhibit anterior cysts and destabilized cloacal cilia. PKD2 suppression inactivates CaMK-II in pronephric cells and cilia, whereas constitutively active CaMK-II restores pronephric duct formation in pkd2 morphants. PKD2 and CaMK-II deficiencies are synergistic, supporting their existence in the same genetic pathway. It is concluded that CaMK-II is a crucial effector of PKD2 Ca2+ that both promotes morphogenesis of the pronephric kidney and stabilizes primary cloacal cilia (Rothschild, 2011).

Phosphorylation of Numb family proteins

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), affinity chromatography purification was performed using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), two Numb family proteins (Numb and Numbl) were identified from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl are efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, the phosphorylation of Numb family proteins was observed in various rat tissue extracts, and the ionomycin-induced phosphorylation of endogenous Numb was detected at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, the recruitment of 14-3-3 proteins was found to be the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein (Tokumitsu, 2005).

The role of CaM kinase II in T cell memory

The multifunctional calcium/calmodulin kinase II family consists of at least four different isoforms that mediate part of the cellular response to calcium in various tissues. The alpha and beta isoforms of CaMKII are exclusively expressed in the brain, whereas the gamma and delta isoforms of CaMKII are ubiquitously expressed. In the brain, CaMKII function is intricately linked to the induction of LTP and spatial learning as shown by the analysis of mice that express a calcium-independent form of CaMKIIalpha or mice that are deficient in CaMKIIalpha. There is only a limited understanding of CaMKII function in other tissues. CaMKII can reveal kinase activity in two ways. Upon calcium influx, calcium-calmodulin binds to CaMKII and induces high levels of kinase activity. Activated CaMKII phosphorylates various substrates including threonine 287 for gamma CaMKII (T286 for alpha CaMKII) in the CaMKII inhibitory domain. Autophosphorylation of Thr-287 causes CaMKII subunits to acquire a high affinity for calmodulin in the absence of calcium, such that calmodulin can remain 'trapped' on autophosphorylated CaMKII; this results in autonomous, or calcium-independent, CaMKII activity. CaMKII exists as a holoenzyme composed of 6-12 subunits, and the proportion of autophosphorylated subunits is determined by the frequency of calcium oscillations produced by influx through calcium-selective channels such as the postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors. CaMKII can thus 'store' the previous frequency of calcium oscillations in the form of phosphothreonines such that CaMKII is a biochemical decoder that contributes to at least one aspect of the frequency response function leading to LTP in hippocampal neurons. Conceptually, calcium-dependent activity reflects acute activation, whereas the level of autonomous CaMKII activity reflects the previous frequency of calcium oscillations (Bui, 2000 and references therein).

In immunocompetent T cells, calcium is an important second messenger that contributes to the induction of proliferation, anergy, and cell death. It thus affects acute T cell activation and also long-term changes in antigen responsiveness. As a major downstream effector of calcium-calmodulin in T cells, CaMKII has the biochemical design to regulate activation, anergy, and memory. To determine the effect of CaMKIIgamma on T cells, a calcium-independent mutant of CaMKIIgamma-B has been constructed analogous to the CaMKIIalpha mutant used to study neuronal LTP. This mutant (CaMKIIgammaB) encodes a negatively charged aspartic acid substituted for the threonine 287 phosphorylation site and exhibits37% autonomous activity. In Jurkat T cells, CaMKIIgamma-B* squelches IL-2 production: this was interpreted as an indication that CaMKII may be a mediator of T cell anergy (Bui, 2000 and references therein).

In order to understand how CaMKII regulates T cell physiology, and in particular the long-term generation of anergy or memory, mice were generated expressing the calcium-independent mutant of CaMKIIgammaB (CaMKIIgammaB*) in the T cell lineage. In these mice, the size of the thymus is increased 1.5- to 2-fold, at least in part due to an increase in the lifespan of double-positive (DP) thymocytes. More important, there is an increase in the number of T cells in the secondary lymphoid organs that have acquired an antigen-dependent memory phenotype. These T cells are bonafide memory cells as assessed by a variety of criteria. In addition, T cells from wild-type mice acquire calcium-independent CaMKII activity after several rounds of antigen-stimulated division. It is proposed that CaMKII controls a distinct process of activation-induced cellular differentiation (Bui, 2000).

Thus a point mutation in CaMKIIgammaB causes two readily apparent effects on the physiology of the immune system. Most notably, CaMKIIgammaB* enhances the antigen-dependent formation of T cell memory. This reverse genetics approach has been reinforced by the observation that the activation and induction of cell division in primary cultures of T cells causes an increase in endogenous CaMKIIgamma activity. A second observation, that may or may not be related, is that CaMKIIgammaB* lengthens the DP thymocyte lifespan. It is deduced that the memory phenotype of cells from CaMKIIgammaB* mice, as defined by the expression of CD44, CD62L, and CD45RB cell surface markers, is indicative of memory T cells able to mediate a secondary immune response. (1) The conversion to the memory-phenotype increases with age, similar to that which is seen in wild-type mice. (2) The increase in memory-phenotype cells is specific for cells with the potential to recognize environmental antigens, i.e., cells with a transgenic TCR that express CaMKIIgamma-B* remain naive. (3) Memory-phenotype cells from CaMKIIgammaB* mice express an expanded range of cytokines after stimulation that is indistinguishable from wild-type memory cells. (4) T cells from CaMKIIgammaB* mice exhibit an enhanced recall response to antigen. Thus, in every assay, the effect of CaMKIIgammaB* is to enhance a natural process of proliferation and memory formation (Bui, 2000).

There are two possibilities to explain an increase in the number of memory cells. One relates to the fate of antigen-activated T cells. Such cells either undergo activation-induced cell death or differentiate to form memory cells, and it is possible that CaMKIIgamma regulates this fate decision. A second possibility is that the lifespan of the memory cell could be increased; however, this possibility is not tenable given the recent work showing that memory cells have an indefinite lifespan even in the absence of antigen. Instead, the idea is favored that CaMKII regulates the differentiation into memory cells but not the maintenance of the memory phenotype. In support of this interpretation, there is found to be an increased antigen or superantigen-induced expansion of T cells in culture or in vivo. Moveover, at least part of this increase can be explained by a decrease in activation-induced cell death. The notion is that memory cells arise from dividing effector cells and the level of CaMKII autonomous activity is an important regulator of survival and differentiation at this critical step. Remarkably, this is consistent with the role of CaMKII in the induction but not the maintenance of LTP (Bui, 2000).

In the formation of learning and memory in the brain, CaMKII acts as a decoder of the frequency of calcium oscillations. Higher frequencies result in LTP, and lower frequencies result in long-term depression. This decoding function, and memory itself, are critically dependent on the establishment of autonomous CaMKII kinase activity through the phosphorylation of threonine 286 (Giese, 1998). Is it possible that CaMKII in T cells is regulated in the similar fashion? A body of work has shown that calcium oscillations occur in nonexcitable cells, and both frequency and amplitude can be determined by the characteristics of agonist-receptor interactions. In particular, ligation of the TCR causes calcium oscillations in T cells, and the oscillatory frequency can be the determining factor in activation of transcription factors such as NF-AT and NFkappaB. Furthermore, recent evidence indicates that memory T cells arise from activated T cells that have undergone numerous rounds of activation-induced cell division. It is suggested that the duration of signaling through the TCR and other factors, such as coreceptor stimulation, may affect the frequency and perhaps the amplitude of calcium oscillations. This, in turn, could determine the level of autonomous CaMKII activity and the probability of survival as a memory cell (Bui, 2000).

Although an analogy is drawn between survival and memory in lymphocytes and LTP in neurons, there are most certainly important differences. Intracellular calcium oscillations in lymphocytes result from the release of calcium from intracellular stores and the activation of store-operated calcium (CRAC) channels in the plasma membrane. In contrast, calcium oscillations in postsynaptic cells result from an entirely different mechanism that involves calcium influx through NMDA receptors. In addition, lymphocyte memory involves survival of proliferating cells in the face of activation-induced cell death, whereas LTP occurs in a nondividing neuronal population. It can be imagined that CaMKII is but one processor of cell-type specific input and functions to initiate differentiation programs that are entirely divergent in lymphocytes and neurons. Survival is not sufficient for enhanced memory T cell formation. In mice expressing a Bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue) transgene in the T cell lineage, activation-induced cell death is decreased, but there is no observed increase in the number of memory cells. Yet, whereas Bcl-2 is not sufficient to enhance memory cell formation, it is possible that it is a downstream target of CaMKII (Bui, 2000 and references therein).

An extravagant aspect of T cell development is the massive cell death that occurs as a natural part of thymic selection. Rather than die continuously with a constant probability, thymocytes have a fixed lifespan of approximately 3.5 days, and this fixed lifespan is manifested in thymocyte survival in vitro as well. Thus, there must be a mechanism to keep track of time in this nondividing population. Since CaMKIIgammaB* mice display an extended lifespan both in vivo and in vitro, the calcium-independent form of CaMKIIgamma thus appears to regulate the timing of programmed cell death. This may be a key to its function, both in the thymus and the generation of memory T cells. Again, there are similarities, but also important differences in comparing the thymic phenotypes of Bcl-2 and CaMKIIgammaB transgenic mice. Both double positive and single positive thymocytes from Bcl-2 transgenic mice exhibit an increased lifespan measured in vitro, and double positive thymocytes from Bcl-2 transgenic mice label very slowly with an estimated in vivo lifespan of more than 8 days . Despite this profound effect on survival, Bcl-2 mice do not have an increase in total thymocytes but do show a greater number of single positives and a skewed selection toward the CD8 lineage. It is concluded that, separate from the survival effects of Bcl-2, CaMKII plays a part in the strict control of double postive lifespan. In this regard, it will be worthwhile to dissect how CaMKII may link up to the mechanisms that measure time (Bui, 2000 and references therein).

CaM kinase II in left-right asymmetry

Intracellular calcium ion [Ca(2+)] elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca(2+)/CaM-dependent protein kinase (CaMK-II) is identified as a necessary target of this Ca(2+) elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six- and 12-somite stages, which is coincident with known left-sided Ca(2+) elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (alphaKAP) that can hetero-oligomerize with and target active enzyme to membranes. alphaKAP, beta2 CaMK-II and gamma1 CaMK-II antisense morpholino oligonucleotides, as well as KV-targeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca(2+) channel and gamma1 CaMK-II; however, alphaKAP, beta2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca(2+)-sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca(2+) signals to the LPM (Francescatto, 2010).

CaM kinase in neurons

Calcium/calmodulin-dependent protein kinase II (CaMK II) and p42 mitogen-activated protein kinase (MAPK) (Drosophila homolog:Rolled Rolled) are enriched in neurons and possess the capacity to become persistently active, or autonomous, following removal of the activating stimulus. Since persistent kinase activation may be a mechanism for information storage, primary cultures of cortical neurons were used to investigate whether kinase autonomy can be triggered by bursts of spontaneous synaptic activity. Both kinases respond to synaptic stimulation, but differ markedly in their kinetics of activation and inactivation, as well as in their sensitivity to NMDA receptor blockade. While 90% of maximal CaMK activation is observed after only 10 sec of synaptic bursting, MAPK activity is unaffected at this early time and rises to only 30% of maximal after 2 min of stimulation. Following blockade of synaptic stimulation, CaMK activity decreases by 50% in 10-30 sec, while MAPK activity decays by 50% within 6-10 min. Although MAPK exhibits relatively slow activation, short periods of synaptic activity can trigger the MAPK activation process, which persist in the absence of synaptic stimulation. Comparison of the effect of NMDA receptor blockade on synaptic activation of these kinases reveals that CaMK II activity is preferentially suppressed. Since CaMK II is concentrated in dendritic processes in the vicinity of synapses, synaptic calcium transients were measured in fine dendritic processes (approximately 1 microns diameter) to assess their sensitivity to NMDA receptor blockade. Calcium transients in these fine processes are reduced by up to 90% by NMDA receptor blockade, possibly accounting for the profound sensitivity of CaMK II to this treatment. The sharp contrast between the regulation of CaMK II and MAPK by synaptic activity indicates that they may mediate neuronal responses to different patterns of afferent stimulation. The relatively slow activation and inactivation of MAPK suggests that it may be able to integrate information from multiple, infrequent bursts of synaptic activity (Murphy, 1994).

To investigate the physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, the alpha subunit of mouse CaM kinase II (CaM kinase II alpha) was transfected into PC12 cells and clonal cell lines were established that constitutively express the transfected CaM kinase II alpha gene. Northern blot analysis shows that the gamma and delta subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activates CaM kinase II alpha through autophosphorylation and generation of the Ca2+/calmodulin-dependent form. It is interesting that the neurite outgrowth induced by cyclic AMP is inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase II alpha. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system (Tashima, 1996).

Calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes calcium-dependent autophosphorylation, generating a calcium-independent form that may serve as a molecular substrate for memory. Calcium-independent CaMKII specifically binds to isolated postsynaptic densities (PSDs), leading to enhanced phosphorylation of many PSD proteins including the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptor. Binding to PSDs changes CaMKII from a substrate for protein phosphatase 2A to a protein phosphatase 1 substrate. Translocation of CaMKII to PSDs occurs in hippocampal slices following treatments that induce CaMKII autophosphorylation and a form of long term potentiation. Thus, synaptic activation leads to accumulation of autophosphorylated, activated CaMKII in the PSD. This increases substrate phosphorylation and affects regulation of the kinase by protein phosphatases, which may contribute to enhancement of synaptic strength (Strack, 1997).

The association of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with postsynaptic densities (PSDs) was determined by activity assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting of the enzyme. Soluble CaM kinase II was autophosphorylated with ATP in the presence of Ca2+ and calmodulin, and then it was incubated with PSDs. Autophosphorylated CaM kinase II precipitates with PSDs by centrifugation, but kinase that is not autophosphorylated does not precipitate with PSDs. These results indicate that the soluble previously autophosphorylated CaM kinase II associates with PSDs and forms PSD-CaM kinase II complex. A maximum of about 60 micrograms of soluble CaM kinase II binds to 1 mg of PSD protein under the experimental conditions. Ca2+-independent activity generated by autophosphorylation of the kinase is retained in the PSD-CaM kinase II complex. The CaM kinase II thus associated with PSDs phosphorylates a number of PSD proteins in both the absence and presence of Ca2+. When the CaM kinase II-PSD complex is incubated at 30 degrees C, its Ca2+-independent activity is gradually decreased. This decrease is correlated with dephosphorylation of the kinase and its release from PSD-CaM kinase II complex. These results indicate that CaM kinase II reversibly translocates to PSDs in a phosphorylation-dependent manner (Yoshimura, 1997).

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).

The results presented here support the notion that differential expression of PSD-95-binding proteins in different neurons helps to determine the composition of signal transduction complexes formed by association with PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define the nature of local biochemical signaling associated with activation of NMDA receptors. The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made onto glutamatergic neurons in the hippocampus. CaM kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses will differ dramatically between excitatory and inhibitory neurons. High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus. In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often found together in excitatory neurons. Thus, the composition of signal transduction machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in other areas of the brain (Zhang, 1999).

Calcium-calmodulin-dependent protein kinase II (CaMKII) promotes the maturation of retinotectal glutamatergic synapses in Xenopus. Whether CaMKII activity also controls morphological maturation of optic tectal neurons was tested using in vivo time-lapse imaging of single neurons over periods of up to 5 days. Dendritic arbor elaboration slows with maturation, and correlates positively with the onset of CaMKII expression. Elevating CaMKII activity in young neurons by viral expression of constitutively active CaMKII slows dendritic growth to a rate comparable to that of mature neurons. CaMKII overexpression stabilized dendritic structure in more mature neurons, whereas CaMKII inhibition increases their dendritic growth. Thus, endogenous CaMKII activity limits dendritic growth and stabilizes dendrites, and it may act as an activity-dependent mediator of neuronal maturation. New retinal axonal branches may form transient pure NMDA synapses with tectal neurons, but these branches have no impact on the postsynaptic activity unless they are coactive with other activity in the tectal neuron. If these conditions are met, NMDA receptors are active and result in calcium influx at that synaptic site. Elevated calcium then activates CaKII locally, reflecting the spatial and temporal patterns of afferent coactivity. Elevated CaMKII activity might then promote the maturation of the synapse through the addition of a functional AMPA component to synaptic responses and would locally stabilize tectal cell dendrites (Wu, 1998).

NMDA or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). At short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction. However, 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected; it occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or nitrogen oxide (NO) release (Johnston, 1995).

Neuronal activity is required for normal neural development, however excessive activity can cause abnormal growth of neural processes and may contribute to formation of epileptic foci. PC12 cells were used to investigate mechanisms by which depolarization regulates neurite growth. Depolarization with 45 mM KCl induces neurite outgrowth only if NGF receptors are partly activated by overexpression of p140trkA or by treatment with a low concentration of NGF that alone is insufficient to stimulate neurite formation. Depolarization-induced neurite growth is reduced by inhibitors of L-type Ca2+ channels, Ca2+/calmodulin-dependent protein (CaM) kinases II and IV, and transcription. These results identify a novel mechanism by which depolarizing stimuli synergize with subthreshold activation of NGF receptors to induce neurite growth through a Ca2+ and CaM kinase-dependent signal transduction pathway (Solem, 1995).

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription (Hu, 1999).

Olfactory signal transduction in the olfactory epithelium (OE) is mediated by second messenger cascades. Odorant receptors exhibit structural characteristics of the seven transmembrane G protein-coupled receptor superfamily. The application of rapid kinetics methodology has shown that odorants elicit a rapid elevation of either cAMP or IP3. The colocalization of a unique G protein, Golf, the olfactory adenylyl cyclase (AC3), and cyclic nucleotide-gated (CNG) cation channels in the olfactory cilia suggests an important role of cAMP in olfactory signal transduction. Indeed, a variety of odorants stimulate adenylyl cyclase activity in membrane preparations from OE. Odorant receptor activation increases intracellular cAMP through interactions with Golf. This leads to the opening of CNG cation channels, membrane depolarization, and the generation of action potentials. It was recently discovered that excitatory responses to both cAMP and IP3-producing odorants are undetectable in mice lacking functional olfactory CNG channels, further suggesting that cAMP may be a major second messenger mediating olfactory signaling. Thus, the coupling of olfactory receptors to adenylyl cyclase is a major mechanism for olfactory signal transduction (Wei, 1998 and references).

One of the characteristic features of odorant-induced second messenger signaling is the transient responsiveness to odorant. Rapid increases and subsequent decreases in cAMP or IP3 have been observed both in olfactory cilia and in primary olfactory neuron cultures. In vitro biochemical approaches and studies with transgenic mice have suggested several mechanisms for desensitization, including odorant receptor phosphorylation, activation of PDEs, and ion channel regulation. Although increases in intracellular Ca2+ are thought to terminate olfactory signaling, mechanisms for inhibition of olfactory signaling by Ca2+ are not well defined. Ca2+ influx through the CNG channels may trigger adaptation of odorant responses by targeting multiple steps in the cAMP signaling cascade. For example, olfactory receptor neurons express a Ca2+/calmodulin- (CaM) activated PDE, and PDE inhibitors prolong odor-induced cAMP formation in olfactory cilia. This suggests that a CaM-sensitive PDE may contribute to signal termination. Intracellular Ca2+ also decreases the sensitivity of the CNG channel to cAMP, suggesting that Ca2+-regulated channels may also participate in the adaptation process (Wei, 1998 and references).

AC3 and CaMKII are both expressed in the cilia of olfactory neurons. An immunohistochemical analysis of OE detects AC3 primarily in the apical ciliary layer. There is little or no AC3 detectable in olfactory receptor neuron cell bodies. CaMKII immunoreactivity is also evident in the apical ciliary layer as well as neuron cell bodies. The merged image indicates that AC3 and CaMKII are coexpressed in olfactory cilia, the structures that contain olfactory receptors and are the primary source of olfactory signaling. Therefore, CaMKII has the potential to regulate AC3 activity in olfactory neurons during olfactory signaling, since they are both expressed in the cilia of olfactory sensory neurons (Wei, 1998).

Odorant stimulation of increases in cAMP in the OE is mediated by AC3 (a Ca2+-inhibited adenylyl cyclase). Inhibition of AC3 by intracellular Ca2+ is mediated by CaM kinase II (CaMKII) phosphorylation of AC3 at Ser-1076; the mutation of Ser-1076 to alanine renders AC3 insensitive to Ca2+ inhibition. Since odorant stimulation of intracellular cAMP is accompanied by increased intracellular Ca2+, CaMKII phosphorylation of AC3 may contribute to the cAMP transients in olfactory cilia. To test this hypothesis, a polyclonal antibody specific for AC3 phosphorylated at Ser-1076 was generated. The phosphorylation of AC3 at Ser-1076 is significantly enhanced upon stimulation with an odorant, and this phosphorylation is mediated by CaMKII. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulates phosphorylation of AC3 at Ser-1076. To determine what fraction of olfactory neurons responds to odorants, cultured olfactory neurons were treated with citralva or isoamyl alcohol (IA) for 30 s and immunostained for phosphorylated AC3. Only 2% ± 0.4% of the cells show a detectable signal in the absence of odorant, whereas 11.0% ± 1.6%, 40% ± 7.0%, and 91% ± 9.2% of the cells are positive for phosphorylated AC3 when olfactory neurons are treated with IA, citralva, or forskolin, respectively. The high percentage of cells activated by citralva is not unexpected; citralva is a complex mixture of odorants that undoubtedly stimulates multiple receptors in olfactory cilia. Phosphorylation of AC3 is blocked by inhibitors of CaMKII, which also ablates cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses (Wei, 1998).

Olfactory signal transduction involves the activation of a G-protein-coupled adenylyl cyclase/cAMP second messenger cascade leading to the sequential opening of Ca2+-permeable cAMP-gated cation channels and Ca2+-activated chloride channels. Like sensory neurons of other modalities, vertebrate ORNs adapt to ambient conditions by time-dependent modification in the sensitivity to a given stimulus, as seen in the decline of the sensory response during prolonged odor stimulation. Odor adaptation in vertebrate olfactory receptor neurons (ORNs) is commonly attributed to feedback modulation caused by Ca2+ entry through the transduction channels, but it remains unclear and controversial whether this Ca2+-mediated adaptation resides in the cAMP-gated channel alone or whether other molecules of the transduction cascade are modulated as well. Attenuation of adenylyl cyclase activity by Ca2+/calmodulin-dependent protein kinase II (CaMKII) has also been proposed as a mechanism for adaptation. To test this in intact ORNs, the properties of adaptation induced by a sustained (8 sec) or brief (100 msec) odor stimulus were compared. Although adaptation induced by both types of stimuli occurs downstream from the odor receptors and is Ca2+-dependent, only adaptation induced by a sustained pulse involves alterations in the odor response kinetics, consistent with a reduction in the rate of adenylyl cyclase activation. By disrupting CaMKII to block adenylyl cyclase attenuation using a specific peptide inhibitor of CaMKII, autocamtide-2-related inhibitory peptide (AIP), it has been shown that this reaction is necessary for odor adaptation in vivo. With CaMKII disrupted, adaptation induced by a sustained stimulus is significantly impaired: the onset rate of adaptation is decreased by threefold, and the recovery rate from adaptation is increased by up to sixfold. In contrast, adaptation induced by a brief odor pulse is unaffected, demonstrating that the effect of AIP must be highly specific. The results indicate that CaMKII controls the temporal response properties of ORNs during odor adaptation. It is proposed that CaMKII plays a prominent role in odor perception (Leinders-Zufall, 1999).

The regulation of AMPA receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC) was studied. Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1A receptor agonists (see Drosophila Serotonin receptor 1A) and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1A agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. It is concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice (Cai, 2002).

The serotonin system and NMDA receptors (NMDARs) in prefrontal cortex (PFC) are both critically involved in the regulation of cognition and emotion under normal and pathological conditions; however, the interactions between them are essentially unknown. Serotonin, by activating 5-HT1A receptors, inhibits NMDA receptor-mediated ionic and synaptic currents in PFC pyramidal neurons, and the NR2B subunit-containing NMDA receptor is the primary target of 5-HT1A receptors. This effect of 5-HT1A receptors is blocked by agents that interfere with microtubule assembly, as well as by cellular knock-down of the kinesin motor protein KIF17 (kinesin superfamily member 17), which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Inhibition of either CaMKII (calcium/calmodulin-dependent kinase II) or MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) abolished the 5-HT1A modulation of NMDAR currents. Biochemical evidence also indicates that 5-HT1A activation reduced microtubule stability, which was abolished by CaMKII or MEK inhibitors. Moreover, immunocytochemical studies show that 5-HT1A activation decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Together, these results suggest that serotonin suppresses NMDAR function through a mechanism dependent on microtubule/kinesin-based dendritic transport of NMDA receptors that is regulated by CaMKII and ERK signaling pathways. The 5-HT1A-NMDAR interaction provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes subserved by PFC (Yuen, 2005).

Synaptic vesicle availability and mobilization are important parameters in the regulation of synaptic transmission and synaptic plasticity. Synapsins, a family of highly conserved neuronal phosphoproteins that are specifically associated with synaptic vesicles, have been implicated in the regulation of neurotransmitter release by controlling the number of vesicles available for exocytosis. Synapsins exist in all organisms with a nervous system, and are encoded by three distinct genes, synapsin I, II, and III, in most vertebrates and by a single gene, Synapsin, in Drosophila. Synapsins are the most abundant synaptic vesicle proteins, with synapsin I alone accounting for 6% of total vesicle protein. They are present in nearly all presynaptic nerve terminals, but different neurons have distinct repertoires of different synapsins (Chi, 2003 and references therein).

During action potential firing, the rate of synapsin dissociation from synaptic vesicles and dispersion into axons controls the rate of vesicle availability for exocytosis at the plasma membrane. Synapsin Ia's dispersion rate tracks the synaptic vesicle pool turnover rate linearly over the range 5-20 Hz: the molecular basis for this lies in regulation at both the calcium-calmodulin-dependent kinase (CaM kinase) and the mitogen-activated protein (MAP) kinase/calcineurin sites in the Synapsin Ia protein. These results show that CaM kinase sites control vesicle mobilization at low stimulus frequency, while MAP kinase/calcineurin sites are critical at both lower and higher stimulus frequencies. These results support a model in which the speed of dissociation of synapsin from synaptic vesicles and redistribution into the axon controls the availability of synaptic vesicles for fusion with the plasma membrane. Thus, multiple signaling pathways serve to allow synapsin's control of vesicle mobilization over different stimulus frequencies (Chi, 2003).

Competitive synaptic remodeling is an important feature of developmental plasticity, but the molecular mechanisms remain largely unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can induce postsynaptic changes in synaptic strength. Postsynaptic CaMKII also generates structural synaptic rearrangements between cultured cortical neurons. Postsynaptic expression of activated CaMKII (T286D) increases the strength of transmission between pairs of pyramidal neuron by a factor of 4, through a modest increase in quantal amplitude and a larger increase in the number of synaptic contacts. Concurrently, T286D reduces overall excitatory synaptic density and increases the proportion of unconnected pairs. This suggests that connectivity from some synaptic partners increases while other partners are eliminated. The enhancement of connectivity requires activity and NMDA receptor activation, while the elimination does not. These data suggest that postsynaptic activation of CaMKII induces a structural remodeling of presynaptic inputs that favors the retention of active presynaptic partners (Pratt, 2003).

During CNS development, the initial patterns of connectivity are highly divergent and must be refined through a process of activity-dependent competition for synaptic space on the postsynaptic neuron. For example, early in development LGN neurons receive inputs from >20 retinal ganglion cells, but after a few weeks they are connected to only 1-3. This occurs through a structural remodeling of presynaptic contacts such that some presynaptic partners gain synaptic contacts, while others lose contacts and become entirely disconnected from the postsynaptic neuron. A similar process occurs during the segregation of LGN projections to layer IV of primary visual cortex, the reduction in climbing fiber input onto cerebellar Purkinje neurons, and loss of multiple innervation at the neuromuscular junction. These structural rearrangements in presynaptic connectivity depend upon competition between inputs, because activity in one set of inputs is required to drive the elimination of others. Despite the ubiquity of such structural rearrangements during developmental plasticity, the molecular machinery that allows activity of one presynaptic partner to increase the number of stable synaptic contacts, while eliminating contacts from other inputs, is still unknown (Pratt, 2003).

CaMKII is an attractive candidate to mediate such heterosynaptic structural rearrangements. CaMKII is a serine-threonine kinase that is activated by neuronal activity and can localize to the postsynaptic density. Calcium influx through NMDARs allows calcium/calmodulin to bind to CaMKII, which activates the enzyme. Once activated, CaMKII can autophosphorylate on Thr286, which renders the enzyme calcium independent and capable of phosphorylating other substrates even in the absence of calcium. These properties have led to the suggestion that CaMKII is a molecular switch that is moved into a persistently active state by a transient rise in calcium, which can be driven by correlated pre- and postsynaptic activity (Pratt, 2003).

There is a wealth of evidence that CaMKII is required for activity-dependent synaptic strengthening. CaMKII participates in LTP at central synapses by both phosphorylating synaptic AMPA receptors (AMPARs) and causing insertion of AMPARs into existing synaptic sites. An additional role in structural plasticity has been suggested by studies showing that CaMKII nulls or transgenes compromise activity-dependent cortical plasticity, although how these effects are mediated at the cellular level remains unknown. The ability of postsynaptic CaMKII to stabilize retinotectal dendrites and axons in Xenopus and to regulate synaptic density and synaptic structure in invertebrates suggests that in addition to postsynaptic changes in receptor trafficking, CaMKII could play an important role in regulating the formation or stability of synaptic connections (Pratt, 2003).

This study shows that postsynaptic activation of CaMKII leads to structural rearrangements that enhance connections from some presynaptic partners, while eliminating connections from others. To directly manipulate the levels of activated CaMKII, individual cortical pyramidal neurons were transfected with a peptide inhibitor of CaMKII, or a constitutively active form of CaMKII (T286D) in which the autophosphorlyation at Thr286 was mimicked by changing Thr286 to Asp286. T286D is catalytically active in the absence of calcium and can still form holoenzymes and localize correctly to the PSD. Expressing T286D in the postsynaptic neuron selectively enhanced connectivity from some presynaptic partners. At the same time, T286D produced a net loss of presynaptic contacts and a reduction in the number of presynaptic partners. The loss of presynaptic contacts required only constitutive kinase activity, while the enhancement of connectivity required both constitutive kinase activity and ongoing NMDAR activation. These data suggest that postsynaptic activation of CaMKII induces dramatic structural rearrangements in presynaptic connectivity that favors active presynaptic partners (Pratt, 2003).

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIß, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIß, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIß controlling the dendritic morphology and number of synapses (Fink, 2003).

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase have been shown to provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses (Wen, 2004).

Axon guidance by a number of guidance molecules has been shown to depend on localized Ca2+ signals in the growth cone. Using direct focal photoactivated release of caged Ca2+ in the growth cone, it has been found that a localized Ca2+ signal in the growth cone is sufficient to induce growth cone attraction as well as repulsion, depending on the resting level of intracellular Ca2+ concentrations ([Ca2+]i) at the growth cone. Similarly, studies on netrin-1 guidance have indicated that different Ca2+ signals might underlie distinct turning responses induced by netrin-1 gradients. These results not only demonstrate a crucial role for Ca2+ signals in growth cone guidance but also indicate that complex Ca2+ mechanisms may operate to control distinct growth cone responses to a wide spectrum of external molecules (Wen, 2004 and references therein).

How different turning responses are generated by distinct Ca2+ signals, however, remains unknown. It is conceivable that different local Ca2+ signals, integrated with the baseline level of [Ca2+]i in the growth cone, activate distinct pathways to mediate attraction and repulsion, respectively. The complexity of Ca2+ signaling in axon guidance is further increased by a series of recent studies demonstrating that Ca2+-dependent growth cone responses can be further modulated by the cAMP pathway: elevation of cAMP leads to switching of repulsion to attraction and vice versa. Does the cAMP pathway act upstream to modify the characteristics of Ca2+ signals (local and/or global) or target the downstream effectors of Ca2+ for the switching? It was recently shown that cAMP/cGMP can affect L-type Ca2+ channels to alter intracellular Ca2+ signals induced by netrin-1, thus placing cAMP/cGMP upstream of Ca2+ in mediating bidirectional turning responses. However, whether cAMP also plays a role downstream of Ca2+ signaling in guidance remains to be evaluated. Most importantly, the question of what downstream targets mediate distinct Ca2+-dependent turning behaviors is still unanswered. Do attraction and repulsion involve the same or separate downstream signaling cascades? In this study, a direct local Ca2+ elevation approach was used to study downstream mechanisms of Ca2+-dependent bidirectional turning responses of nerve growth cones. The use of direct local elevation of intracellular Ca2+ concentrations through photoactivated release of caged Ca2+ bypasses membrane receptor activation and can largely avoid crosstalk among different signaling pathways, thus allowing a focus on intracellular Ca2+ and its downstream events during distinct turning responses. Evidence suggests that Ca2+-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN)-phosphatase-1 (PP1) mediate attraction and repulsion, respectively. Significantly, CaMKII/CaN-PP1 acts as a bimodal switch to control the direction of growth cone turning in response to different Ca2+ signals (local and global) by preferentially activating one component over the other. It is further shown that the cAMP pathway negatively regulates the CaN-PP1 side of the switch to modulate growth cone responses. Finally, evidence is presented that the CaMKII/CaN-PP1 mechanism also mediates netrin-1 guidance. These findings thus provide significant insights toward the downstream mechanisms underlying various turning behaviors induced by complex Ca2+ signals (Wen, 2004).

Small conductance Ca2+-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. In addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels (Bildl, 2004).

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been described as a biochemical switch that is turned on by increases in intracellular calcium to mediate synaptic plasticity. Reductions in CaMKII activity trigger persistent increases in intrinsic excitability. In spontaneously firing median vestibular nucleus (MVN) neurons, CaMKII activity is near maximal, and blockade of CaMKII activity increases excitability by reducing BK-type calcium-activated potassium currents. Firing rate potentiation, a form of plasticity in which synaptic inhibition induces long-lasting increases in excitability, is occluded by prior blockade of CaMKII and blocked by addition of constitutively active CaMKII. Reductions in CaMKII activity are necessary and sufficient to induce firing rate potentiation and may contribute to motor learning in the vestibulo-ocular reflex (Nelson, 2005).

What are the molecular targets of CaMKII, and how do they regulate excitability? Previous studies have identified sodium currents, calcium and potassium currents, and calcium-activated potassium currents in the generation and shaping of action potentials in MVN neurons. Evidence presented here indicates that CaMKII positively regulates BK-type calcium-activated potassium currents. CaMKII may phosphorylate BK channels directly, leading to net increases in BK currents. A direct regulation of BK channels by CaMKII has been found in human mesangial cells and at the Drosophila neuromuscular junction (Zhou, 1999). In hippocampal neurons, CaMKII phosphorylation of Kv4.2 channel subunits regulates excitability in an activity-dependent fashion. Chronic inhibition of CaMKII results in increased firing in cultured hippocampal neurons, but the underlying mechanisms are not known. CaMKII could regulate BK channels indirectly, via their calcium sources, which in MVN neurons are likely to include T-type and R-type calcium channels. Recent studies have demonstrated that the voltage dependence of T-type channel activation can be shifted to more hyperpolarized potentials by CaMKII phosphorylation. If decreases in CaMKII activity led to dephosphorylation of T-type channels in MVN neurons, a corresponding shift in channel activation would reduce calcium availability for BK channels, resulting in increased neuronal excitability (Nelson, 2005).

Vestibular nucleus neurons play a key role in motor learning in the vestibulo-ocular reflex (VOR). MVN neurons transform head motion signals into the appropriate oculomotor commands. Under conditions that led to motor learning in the VOR, MVN neurons exhibited dramatic changes in their firing responses during head movements that mediate improvements in oculomotor performance (Lisberger and Pavelko, 1988). Motor learning induced by vestibular dysfunction is accompanied by increases in the intrinsic excitability of MVN neurons. Regulation of CaMKII activity and firing rate potentiation could play a critical role. In the intact system, high firing rates of MVN neurons maintain CaMKII in an elevated state. Peripheral dysfunction disrupts excitatory drive from the vestibular nerve and results in acute loss of spontaneous firing rates in MVN neurons in vivo. A sustained reduction in firing rate and intracellular calcium levels would trigger FRP: CaMKII activity would decrease, leading to a reduction in BK currents and increases in excitability. Reductions in CaMKII activity could also modulate synaptic receptors and may account for the decreases in GABA receptor sensitivity observed after motor learning (Nelson, 2005).

These findings imply that the rules for calcium-dependent plasticity differ substantially in spontaneously firing versus silent neurons. Although neurons that are silent in vitro have been the overwhelming focus of studies of cellular plasticity, neurons in many systems fire spontaneously in vivo, either intrinsically or via active excitatory synaptic connections. Tonically firing neurons are likely to have elevated levels of intracellular calcium and activation of calcium-dependent signaling molecules, including CaMKII. Plasticity in such neurons could be mediated through synaptic inhibition and decreases in CaMKII activation (Nelson, 2005).

Angelman syndrome (AS) is a severe neurological disorder characterized by mental retardation, motor dysfunction and epilepsy. AS is caused by loss of function of imprinted genes on human chromosome 15q11-13 or by mutations in the UBE3A gene (see Drosophila Ube3a), which resides in this region. Imprinting of this gene results in exclusive expression of the maternal allele in hippocampal neurons and cerebellar Purkinje cells. The UBE3A gene encodes the ubiquitin protein ligase E3A, also known as E6-AP, but its role remains elusive. Heterozygous mice with a maternally inherited Ube3a mutation (here called 'AS mice') show seizures and motor and cognitive abnormalities similar to the symptoms of AS individuals. Biochemical analysis of these mice indicates that calcium/calmodulin-dependent kinase type 2 (CaMKII) activity is reduced and that phosphorylation of the inhibitory Thr305 and Thr306 site is increased. Because increased inhibitory phosphorylation of CaMKII has a severe impact on neuronal function, whether the increased phosphorylation is directly responsible for the major deficits seen in AS was investigated. Female AS mice were crossed with heterozygous males that carried the targeted αCaMKII-T305V/T306A mutation, which prevents inhibitory phosphorylation of αCaMKII (designated as CaMKII-305/6+/- mice. This resulted in offspring with four genotypes: wild-type mice, AS mutants, αCaMKII-305/6+/- mutants and AS/CaMKII-305/6+/- double mutants. Western blot analysis of hippocampal lysates showed a significant increase of Thr305 phosphorylation in AS mice. In contrast, inhibitory phosphorylation was significantly reduced in CaMKII-305/6+/- mutants (40% and in AS/CaMKII-305/6+/- double mutants. Moreover, the decreased kinase activity of the AS mutants was restored to near wild-type levels in the AS/CaMKII-305/6+/- double mutants. Thus, these mutants are suitable for determining the extent to which increased inhibitory phosphorylation of CaMKII underlies the neurological phenotype of AS mice. This study shows that the molecular and cellular deficits of an AS mouse model can be rescued by introducing an additional mutation at the inhibitory phosphorylation site of αCaMKII. Moreover, these double mutants no longer show the behavioral deficits seen in AS mice, suggesting that these deficits are the direct result of increased inhibitory phosphorylation of αCaMKII (van Woerden, 2007).

Activity-dependent CREB (see Drosophila CrebB) phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at voltage gated CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. This study reports a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin (see Drosophila Calmodulin) from the surface membrane to the nucleus. This study shows that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN (see Drosophila Calcineurin) triggers its nuclear translocation. Both betaCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders (Ma, 2014).

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. This study shows that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIalpha weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of CLOCK and BMAL1. These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities (Kon, 2014).

A CaMKII-NeuroD signaling pathway specifies dendritic morphogenesis

The elaboration of dendrites is fundamental to the establishment of neuronal polarity and connectivity, but the mechanisms that underlie dendritic morphogenesis are poorly understood. The genetic knockdown of the transcription factor NeuroD in primary granule neurons including in organotypic cerebellar slices profoundly impairs the generation and maintenance of dendrites while sparing the development of axons. NeuroD mediates neuronal activity-dependent dendritogenesis. The activity-induced protein kinase CaMKII catalyzes the phosphorylation of NeuroD at distinct sites, including endogenous NeuroD at Ser336 in primary neurons, and thereby stimulates dendritic growth. These findings uncover an essential function for NeuroD in granule neuron dendritic morphogenesis. This study also defines the CaMKII-NeuroD signaling pathway as a novel mechanism underlying activity-regulated dendritic growth that may play important roles in the developing and mature brain (Gaudillière, 2004).

NeuroD is expressed in differentiated neurons in a number of brain regions and is particularly high in cerebellar granule neurons. Focus was placed on the potential role of NeuroD in granule neuron morphogenesis. Granule neurons are generated within the external granular layer (EGL) of the cerebellar cortex, where they extend axons that continue to grow even as these neurons migrate to the internal granular layer (IGL). Well after the birth of axons and the formation of parallel fibers, granule neurons elaborate dendrites within the IGL that form the receiving end of synapses with the axon terminals of mossy fibers. Importantly, the sequential generation of granule neuron axons and dendrites in vivo is faithfully recapitulated in primary cultures of rat or mouse cerebellar granule neurons. For this reason and because granule neurons in primary culture display distinct and easily identifiable axons and dendrites, these neurons provide a robust system for the study of axonal and dendritic development (Gaudillière, 2004).

To determine the role of NeuroD in the specification of neuronal morphogenesis, a DNA template-based RNA interference method was used to acutely knock down the expression of NeuroD in differentiated cerebellar granule neurons. A major advantage of the genetic knockdown method is that it allows the assessment of NeuroD function in granule neurons past the stages of granule cell precursor differentiation, in which NeuroD appears to play an essential prosurvival role. Primary cerebellar granule neurons, isolated from postnatal day 6 rat pups and cultured for 2 days (P6+2DIV), were transfected with the U6/nd1 plasmid encoding NeuroD hairpin RNAs (hpRNAs) or the control U6 plasmid, together with a plasmid encoding ß-galactosidase or a plasmid encoding green fluorescent protein (GFP). Granule neurons were fixed four days after transfection and subjected to immunocytochemistry using a mouse monoclonal antibody to ß-galactosidase or a rabbit antibody to GFP. Axons and dendrites of the ß-galactosidase- or GFP-positive transfected neurons were identified based on their morphology and by the expression of the axonal marker Tau and the somato-dendritic marker MAP2. Strikingly, although the NeuroD hpRNA-expressing neurons displayed robust axons, these neurons harbor either profoundly deficient or no dendrites, with the MAP2 signal only evident in the cell body of the NeuroD hpRNA-expressing neurons. By contrast, the control U6-transfected granule neurons exhibited a normal morphological appearance with both robust axons and MAP2-positive dendrites. NeuroD RNAi did not lead to the redistribution of the dendritic and axonal proteins (MAP2 and Tau) in granule neurons which were analyzed each day for 6 days following transfection. Together, these results reveal that the genetic knockdown of NeuroD in granule neurons triggers the loss of dendrites (Gaudillière, 2004).

The characterization of the CaMKII-NeuroD signaling pathway in dendritic growth points to the versatility of CaMKII in the control of neuronal morphogenesis. CaMKII is thought to act locally within dendrites to mediate activity regulation of growth and stabilization of dendrites and dendritic spines. The findings of this study suggest that CaMKIIalpha or a CaMKII heteromer containing the alpha isoform is also employed in neurons in the propagation of a signal from L-type voltage-sensitive calcium channels to NeuroD, thereby orchestrating a program of gene expression leading to dendritic growth and maintenance. Taken together, these studies suggest that CaMKII represents a nodal point in the regulation of both local and transcription-mediated mechanisms of activity-dependent dendritic development (Gaudillière, 2004).

While the CaMKII-induced phosphorylation of NeuroD is required for activity-dependent dendritic growth, it remains to be determined if the phosphorylation of NeuroD at the CaMKII sites is sufficient to induce dendritic growth. The expression of NeuroD or ND-RES (NeuroD designed to be resistant to RNA interference), respectively, in granule neurons deprived of neuronal activity or in membrane-depolarized granule neurons, in which CaMKIIalpha RNAi was triggered, failed to induce dendritic growth. These results suggest that NeuroD expression solely is not sufficient to promote the generation of dendrites, but leave open the possibility that the phosphorylation of NeuroD at the CaMKII sites might be sufficient to induce dendritic growth. Another remaining question for future studies is how the CaMKII-induced phosphorylation of NeuroD regulates the transcriptional function of NeuroD in neurons. Mutation of serines 290 and 336 has little effect on the ability of the transactivation domain of NeuroD to activate transcription of the G4-luc reporter gene. These results raise the possibility that CaMKII-induced phosphorylation regulates aspects of NeuroD function other than transactivation per se, such as the subcellular localization of NeuroD or the binding of NeuroD to DNA. An alternative possibility is that the CaMKII sites of phosphorylation are critical for transcriptional activation, but their importance can only be revealed in the context of an endogenous target gene with surrounding chromatin rather than in the context of a G4-luc reporter gene (Gaudillière, 2004).

Although NeuroD is highly expressed in specific regions of the brain, the NeuroD subfamily of proneural bHLH transcription factor that includes NeuroD2 has a wider pattern of expression including the hippocampus and cerebral cortex. Therefore, an attractive hypothesis is that NeuroD and NeuroD-related factors may generally regulate dendritogenesis in other regions of the mammalian brain beyond the cerebellum. Consistent with the idea that neuronal activity might regulate NeuroD-related proteins, a novel consensus sequence of CaMKII phosphorylation was identified that by database screening is found in a number of brain-enriched transcriptional regulators including NeuroD2. It will be interesting to determine the functional consequences of potential CaMKII-regulation of these transcription factors and in particular that of NeuroD2 (Gaudillière, 2004).

The expression of the NeuroD subfamily in the brain persists well into maturity, suggesting that these proteins may continue to regulate the growth and refinement of dendritic morphology in the adult brain. In light of these observations, the identification of NeuroD as a calcium-regulated transcription factor raises the interesting possibility that NeuroD might serve roles in such processes as synaptic remodeling and plasticity that are critical for the adaptive functions of the brain (Gaudillière, 2004).

Table of contents

CaM kinase II: Biological Overview | Evolutionary Homologs part 1/2 | Regulation | Developmental Biology | Effects of Mutation | References

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