Ca2+/calmodulin-dependent activation and inactivation mechanisms of alphaCaMKII and phospho-Thr286-alphaCaMKII

Thr(286) autophosphorylation is important for the role of alphaCaMKII in learning and memory. Phospho-Thr(286)-alphaCaMKII has been described to have two types of activity: Ca(2+)-independent partial activity and Ca(2+)/calmodulin-activated full activity. We investigated the mechanism of switching between the two activities in order to relate them to the physiological functioning of alphaCaMKII. Using a fluorometric coupled enzyme assay and smooth muscle myosin light chain (MLC) as substrate, we found that (1) Ca(2+)-independent activity of phospho-Thr(286)-alphaCaMKII represents 5.0 (+/-3.7)% of the activity measured in the presence of optimal concentrations of Ca(2+) and calmodulin and (2) Ca(2+) in the presence of calmodulin activates the enzyme with a K(m) of 137 (+/-56) nM and a Hill coefficient n = 1.8 (+/-0.3). In contrast, unphosphorylated alphaCaMKII has a K(m) for Ca(2+) in the presence of calmodulin of 425 (+/-119) nM and a Hill coefficient n = 5.4 (+/-0.4). Thus, the activity of phospho-Thr(286)-alphaCaMKII is essentially Ca(2+)/calmodulin dependent with MLC as substrate. In physiological terms, our data suggest that alphaCaMKII is only activated in stimulated neurones whereas Ca(2+)/calmodulin activation of phospho-Thr(286)-alphaCaMKII can occur in resting cells (approximately 100 nM [Ca(2+)]). Stopped-flow experiments using Ca(2+)/TA-cal [Ca(2+)/2-chloro-(epsilon-amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin] showed that at 100 nM [Ca(2+)] partially Ca(2+)-saturated Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complexes existed. These are likely to account for the activity of the phospho-Thr(286)-alphaCaMKII enzyme at resting [Ca(2+)]. Ca(2+) dissociation measurements by a fluorescent Ca(2+) chelator revealed that the limiting Ca(2+) dissociation rate constants were 1.5 s(-1) from the Ca(2+)/cal.alphaCaMKII and 0.023 s(-1) from the Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complex, accounting for the differences in the Ca(2+) sensitivities of the Ca(2+)/cal.alphaCaMKII and Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII enzymes.     

A model for molecular mechanisms of synaptic competition for a finite resource

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) undergoes Ca(2+)/calmodulin-dependent autophosphorylation of threonine-286/287 (Thr(286/287)). Extremely high concentration of CaMKII in the postsynaptic spine indicates that increase in the Ca(2+) concentration in the spine induced by synaptic activation results in Thr(286/287) autophosphorylation of this enzyme. It has recently been shown that the K(d) value of CaMKII for Ca(2+)/calmodulin (Ca(2+)/CaM) drastically decreases after Thr(286/287) autophosphorylation. Therefore, Ca(2+)/CaM associated with CaMKII becomes tightly bound to this kinase after Thr(286/287) autophosphorylation. This has been called 'Ca(2+)/CaM trapping'. We discussed the functional significance of Ca(2+)/CaM trapping in the neuronal system by a mathematical-modelling approach. We considered neighbouring synapses formed on the same dendrite and different increase in the Ca(2+) concentration in each spine. CaMKII undergoing Thr(286/287) autophosphorylation in each spine is eager to recruit nearby calmodulin in the dendrite for Ca(2+)/CaM trapping. Since the amount of calmodulin is limited, recruiting calmodulin to each spine causes competition among synapses for this finite resource. The results of our computer simulation show that this competition leads to 'winner-take-all': almost all calmodulin is taken by a few synapses to which relatively large increases in the Ca(2+) concentration are assigned. These results suggest a novel form of synaptic encoding of information.     

Dual effect of ATP in the activation mechanism of brain Ca(2+)/calmodulin-dependent protein kinase II by Ca(2+)/calmodulin.

The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [T?r?k, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.     

Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities

Autophosphorylation of Ca(2+)-calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG-tagged wild type and phospho- and dephospho-mimic mutants of alpha-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca(2+)-calmodulin dependent or autonomous kinase activity of recombinant alpha-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased alpha-CaMKII binding to subcellular fractions enriched in post-synaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho-Thr253-CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell.     

Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.     

Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.     

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

Dysregulated phosphorylation of Ca(2+) /calmodulin-dependent protein kinase II-α in the hippocampus of subjects with mild cognitive impairment and Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder and the most prevalent senile dementia. The early symptom of memory dysfunction involves synaptic loss, thought to be mediated by soluble amyloid-beta (Aβ) oligomers. These aggregate species target excitatory synapses and their levels correlate with disease severity. Studies in cell culture and rodents have shown that oligomers increase intracellular calcium (Ca(2+)), impairing synaptic plasticity. Yet, the molecular mechanism mediating Aβ oligomers' toxicity in the aged brain remains unclear. Here, we apply quantitative immunofluorescence in human brain tissue from clinically diagnosed mild cognitive impaired (MCI) and AD patients to investigate the distribution of phosphorylated (active) Ca(2+) /calmodulin-dependent protein kinase-α (p(Thr286)CaMKII), a critical enzyme for activity-dependent synaptic remodeling associated with cognitive function. We show that p(Thr286)CaMKII immunoreactivity is redistributed from dendritic arborizations to neural perikarya of both MCI and AD hippocampi. This finding correlates with cognitive assessment scores, suggesting that it may be a molecular read-out of the functional deficits in early AD. Treatment with oligomeric Aβ replicated the observed phenotype in mice and resulted in a loss of p(Thr286)CaMKII from synaptic spines of primary hippocampal neurons. Both outcomes were prevented by inhibiting the phosphatase calcineurin (CaN). Collectively, our results support a model in which the synaptotoxicity of Aβ oligomers in human brain involves the CaN-dependent subcellular redistribution of p(Thr286)CaMKII. Therapies designed to normalize the homeostatic imbalance of neuronal phosphatases and downstream dephosphorylation of synaptic p(Thr286)CaMKII should be considered to prevent and treat early AD.     

Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations: a simple model

The rules that govern the activation and autophosphorylation of the multifunctional Ca2+-calmodulin kinase II (CaMKII) by Ca2+ and calmodulin (CaM) are thought to underlie its ability to decode Ca2+ oscillations and to control multiple cellular functions. We propose a simple biophysical model for the activation of CaMKII by Ca2+ and calmodulin. The model describes the transition of the subunits of the kinase between their different possible states (inactive, bound to Ca2+-CaM, phosphorylated at Thr(286), trapped and autonomous). All transitions are described by classical kinetic equations except for the autophosphorylation step, which is modeled in an empirical manner. The model quantitatively reproduces the experimentally demonstrated frequency sensitivity of CaMKII [Science 279 (1998) 227]. We further use the model to investigate the role of several characterized features of the kinase--as well as some that are not easily attainable by experiments--in its frequency-dependent responses. In cellular microdomains, CaMKII is expected to sense very brief Ca2+ spikes; our simulations under such conditions reveal that the enzyme response is tuned to optimal frequencies. This prediction is then confirmed by experimental data. This novel and simple model should help in understanding the rules that govern CaMKII regulation, as well as those involved in decoding intracellular Ca2+ signals.     

Lobe-specific functions of Ca2+·calmodulin in alphaCa2+·calmodulin-dependent protein kinase II activation

N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

Dual mechanism of a natural CaMKII inhibitor

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.     

Mechanism of the T286A-mutant alphaCaMKII interactions with Ca2+/calmodulin and ATP

The role of adenosine 5'-triphosphate (ATP) in the activation mechanism of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) was investigated using the T286A non-autophosphorylatable mutant of alphaCaMKII. Characterization of the T286A-alphaCaMKII mutant revealed k(cat) = 0.06 +/- 0.02 s(-1) for the T286A mutant, a 6 (+/- 2)-fold lower value compared to wild-type alphaCaMKII with 100 microM smooth muscle myosin light chain (MLC) as substrate. MLC phosphorylation by the T286A mutant and wild-type alphaCaMKII was cooperative, with Hill coefficients 2.3 +/- 0.1 and 2.4 +/- 0.3, respectively. K(m) values for MLC were 96 +/- 28 microM with T286A-alphaCaMKII and 49 +/- 29 microM for wild-type alphaCaMKII. Thus, while the activity of alphaCaMKII was sensitive to mutation of the Thr(286) residue to Ala, the mechanisms of the wild-type and T286A mutant enzyme appeared similar. K(d) for Ca(2+)/calmodulin was 2-fold reduced to 40 nM compared to that of wild-type alphaCaMKII (75 nM). ATP induced a 9-fold stabilization of Ca(2+)/calmodulin binding to the T286A mutant enzyme. Fluorescence stopped-flow kinetic experiments revealed that two Ca(2+)/calmodulin-enzyme complexes were formed, the first, unaffected by ATP, with association and dissociation rate constants of 2 x 10(7) M(-1) s(-1) and 5 s(-1), respectively, containing calmodulin in extended conformation. The second complex, in which calmodulin adopted a compact conformation, was formed with association rate constant 3 x 10(6) M(-1) s(-1) and dissociation at 0.15 s(-1) in the absence and 0.015 s(-1) in the presence of ATP. These data show that ATP is involved in the activation mechanism by forming two classes of Ca(2+)/calmodulin.alphaCaMKII.ATP complex. It is likely that only one of the complexes is on the activation pathway.     

L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.     

Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.     

Switching characteristics of a model for biochemical-reaction networks describing autophosphorylation versus dephosphorylation of Ca2+/calmodulin-dependent protein kinase II

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been suggested to participate in various cellular phenomena triggered by Ca2+ signalling. In the present study, we addressed the functional role of CaMKII in molecular-signal transduction in cells by mathematical modelling of putative biochemical-reaction networks thought to represent an essential part of molecular events responsible for CaMKII-related cellular phenomena. These networks include Ca2+/calmodulin-dependent threonine-286/287 (Thr286/287) autophosphorylation of CaMKII versus dephosphorylation of the enzyme. Computer simulation of the model was performed to examine the relation between the Ca2+-signalling pattern as an input and the resulting degree of Thr286/287 autophosphorylation (m) as an output. Under the simplified condition that the Ca2+ concentration during Ca2+ signalling was set to remain constant with time, the biochemical-reaction networks were shown to function as a switch. There is a threshold for gamma, a parameter representing the probability that the Thr286/287-dephosphorylated CaMKII subunit binds with the Ca2+/calmodulin complex; if gamma is above this threshold, m increases with time to a large degree (switch-on); otherwise, it remains near zero (switch-off). Mathematically, this sharp onset of m at the threshold can be accounted for by a change in the structure of the dynamic system describing the model, from bistability to monostability; this is analogous to the first-order phase transition in statistical physics. For the oscillatory time course of [Ca2+], switching characteristics were also shown with respect to the frequency and the maximum amplitude of the oscillation. These results suggest that graded information mediated by Ca2+ signalling is digitized into all-or-non information mediated by Thr286/287 autophosphorylation of CaMKII.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.