Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)*

Background: Ca2+-independent autonomous CaMKII activity and nitric oxide (NO) signaling regulate neuronal function and death. Results: NO generated autonomous CaMKII activity by Ca2+/CaM-dependent S-nitrosylation, and CaMKII inhibition protected from NO-induced neuronal cell death. Conclusion: NO-mediated regulation of CaMKII contributes to its pathological functions. Significance: S-Nitrosylation is a novel path to CaMKII autonomy that connects Ca2+- and NO signaling. Both signaling by nitric oxide (NO) and by the Ca2+/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca2+-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca2+/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca2+. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.

GFP-CaMKII fusion proteins were used (25), which were purified after expression in HEK cells. Briefly, HEK cells were homogenized with a motorized pellet pestle (Kontes) for 10 s in 0.4 ml of ice-cold 50 mM PIPES pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM DTT, and complete protease inhibitor (Roche), then centrifuged at 16,000 ϫ g for 20 min. The CaMKII concentration from the resulting supernatant was determined by their GFP-fluorescence measured in a spectrophotometer (Fluoromax 3; Horiba Jobin Yvon). For the comparison of CaMKII wt to T286A, these extracts were adjusted for equal CaMKII and total HEK cell protein amounts (26,27). All other mutants were further purified as previously described (16,24). Protein phosphatase 2A C subunit was purchased from Cayman Chemical. The substrate peptide Syntide-2 was from Genescript.
NO Release-NO release from the NO donor Diethylamine NONOate (DEA-NONOate) was measured spectrophotometrically in reaction buffer or deionized water by the disappearance of absorption at 250 nm due to loss of parent compound (⑀ ϭ 6500 M Ϫ1 cm Ϫ1 ).
CaMKII Autophosphorylation, Nitrosylation, and Oxidation-CaMKII was made autonomous by reacting 200 -300 nM kinase for 5 min with Ca 2ϩ /CaM (1 mM/1 M) and 50 mM PIPES pH 7.1 in the presence of either ATP/Mg 2ϩ (0.1/10 mM) on ice (for Thr-286 autophosphorylation) or at room temperature with DEA-NONOate (3 mM; for nitrosylation; Cayman Chemical) or H 2 0 2 (3 mM; for oxidation). The reaction was diluted and Ca 2ϩ chelated with EDTA (5 mM). Where indicated, kinase was reacted with peroxynitrite (ONOO Ϫ Cayman Chemical) instead of NO, and/or in the absence or presence of the ONOO Ϫ scavengers tryptophan (Sigma) or MnTMPyP (A.G. Scientific).
For experiments where kinase was both phosphorylated and nitrosylated, CaMKII was reacted with DEA-NONOate at room temperature followed by phosphorylation of Thr-286 by addition of ATP/Mg 2ϩ on ice, or this was reversed and kinase was first phosphorylated at Thr-286 on ice, phosphorylation was stopped by addition of EDTA, then the kinase was reacted with NO at room temperature. Thr-305/306 phosphorylation was induced by chelating Ca 2ϩ in the presence of ATP (after Thr-286 phosphorylation or nitrosylation).
CaMKII Activity Assays-Kinase activity was assessed by 32 P incorporation into the peptide substrate syntide 2, either in the presence of Ca 2ϩ /CaM (1.2 mM/1 M) or EGTA (1.5 mM), as previously described (16,24). To ensure that reactions are within the linear range, 1 min reaction times (at 30°C) were used for stimulated activity and Thr-286-induced autonomy (16); reaction times of up to 5 min were used only for conditions with lower activity.
Detection of S-Nitrosylation-Detection of S-nitrosylation was assessed using the biotin-switch method (28). CaMKII WT or mutant was nitrosylated in the presence of Ca 2ϩ /CaM in HEPES pH 7.1 buffer as described above, then diluted in HEN buffer (HEPES pH 7.7, 1 mM EDTA, and 0.1 mM neocuprione). Proteins were then precipitated at Ϫ20°C with 4 volumes of cold acetone for 20 min and centrifuged at 15,000 ϫ g for 10 min. The pellet was rinsed with ice cold acetone, resuspended in HEN buffer, then non-nitrosylated cysteines were blocked with 25 mM MMTS in 2.5% SDS for 30 min at 50°C. Proteins were again acetone precipitated to remove unreacted MMTS and resuspended in HEN buffer with 1% SDS. The nitro-cysteine was then reduced with 1 mM ascorbate and the reduced cysteine labeled using the non-cleavable biotin-BMCC (250 M) for 1 h at room temperature. Unreacted biotin was removed by acetone precipitating the proteins. Proteins were then run on Western blots, and biotinylation was detected using the avidin-based vectastain kit (Vector Laboratories).
Neuronal Cell Death Assays-Neuronal cell death was measured by release of LDH into the media using a cytotoxicity detection kit (Roche) as previously described (6). Briefly, FIGURE 1. Model of CaMKII structure and regulation. A, CaMKII forms 12meric holoenzymes via C-terminal association domains, with the kinase domains radiating outward (1,2). Each kinase subunit is activated separately by direct binding of Ca 2ϩ /CaM, which can also induce intra-holoenzyme inter-subunit Thr-286 autophosphorylation (9). B, structure of the CaMKII kinase (space-fill) and regulatory (ribbon) domains (2). In the basal inactive state, the substrate binding S-site (orange) is blocked by the regulatory domain, which is held in place in part by interactions with the neighboring T-site (yellow). Thr-286 (red) autophosphorylation (8, 9, 14 -16) or T-site-mediated binding to GluN2B (10) generates "autonomous" CaMKII activity, likely by preventing complete re-association of the regulatory domain with the kinase domain (1). The Cys-280 and Cys-289 S-nitrosylation sites identified here are marked in black. C, sequence of the CaMKII␣ autoregulatory domain with specific residues and regions marked as in panel B. The autoregulatory domain of the CaMKII␤, ␥, and ó isoforms is highly homologous, with only one amino acid exchanged (underlined). The Met-281/282 oxidation (11) and Ser-280 glycosylation (12) sites that also generate CaMKII autonomy are indicated. Note that all pathways to CaMKII autonomy require an initial Ca 2ϩ / CaM-stimulus, and that even autonomous activity is further stimulated by Ca 2ϩ /CaM (1). medium density primary disassociated hippocampal cultures were prepared from newborn Sprague-Dawley rats and plated onto poly-D-lysine-coated 24-well plates. Cultures were maintained in Neurobasal A media with B-27 supplement, 50 units/ml penicillin/streptomycin and 2 mM Glutamax at 37°C in 5% CO 2 . After 14 DIV, cell death was induced by addition of 100 M glutamate or 300 M DEA-NONOate to the media for 5 min. Wells with inhibitor had 100 M APV or 5 M tatCN21 or scrambled control peptide included during the treatment as well a 20-min pretreatment.

RESULTS
NO Generates Autonomous CaMKII Activity-Incubation of purified CaMKII␣ (in the presence of Ca 2ϩ /CaM) either with an oxidizer (H 2 O 2 ) or with an NO donor (DEA-NONOate) both generated autonomous activity (measured after chelation of Ca 2ϩ ), with higher autonomy generated by the NO donor ( Fig. 2A). NO release by the NO donor is well controlled, with no release under acidic conditions (as in deionized water) and with near maximal release within 5 min after addition to buffer at pH 7.2 ( Fig. 2B). As expected, NO-induced autonomy was abolished by C280/M281V mutation (analogous to CaMKII␦ oxidation and M281/282V mutation; Ref. 11). However, surprisingly, autonomy was also abolished by C289V mutation ( Fig. 2A), indicating that simultaneous S-nitrosylation of both Cys-280 and Cys-289 was required to induce autonomy, and that individual nitrosylation of either residue alone was not sufficient.
NO-induced CaMKII Autonomy Is Independent of Thr-286 and Blocked by tatCN21 but Not KN93-To test the possibility that generation of autonomous CaMKII activity by the NO donor was dependent on promoting Thr-286 autophosphory lation, a CaMKII T286A mutant was used. The NO donor induced autonomy also for the CaMKII T286A mutant (Fig.  2C), indicating that the effect was independent of Thr-286 phosphorylation. If any, NO-induced autonomy of the T286A mutant was even higher compared with CaMKII wild type ( Fig.  2C), an effect that could be due to reduced auto-inhibitory interactions within the T286A mutant. Additionally, the effects of two mechanistically distinct CaMKII inhibitors were tested: KN93, which is Ca 2ϩ /CaM-competitive (29), and tatCN21, which does not act through competition with Ca 2ϩ /CaM (27). As expected based on these different modes of inhibition, tatCN21 completely blocked NO-induced autonomous CaMKII activity, while KN93 had no significant effect (Fig. 2C). The same differential effect of these two inhibitors has been observed previously for Thr-286-induced CaMKII autonomy (6).
Induction of NO-mediated Autonomy Requires an Initial Ca 2ϩ /CaM Stimulus-While a 5-min incubation with the NO donor and Ca 2ϩ /CaM generated autonomous CaMKII activity (Figs. 2A and 3A), it did not affect maximally Ca 2ϩ /CaM-stimulated activity (Fig. 3A), as measured by subsequent activity assays with or without chelation of Ca 2ϩ after the initial incubation. Presence of Ca 2ϩ /CaM was required to induce CaMKII autonomy by the NO donor, as no autonomous activity was induced when Ca 2ϩ /CaM was omitted from the initial incuba-tion (Fig. 3A). Thus, generation of this novel form of CaMKII autonomy requires coinciding NO and Ca 2ϩ signals.
Prolonged Exposure to Nitric Oxide Reduces CaMKII Activity-A previous study showed that NO inhibits CaMKII activity through S-nitrosylation at C6 (30). To address this apparently conflicting finding, CaMKII was exposed to NO for varying periods of time (Fig. 3B). Indeed, while Ca 2ϩ /CaM-stimulated CaMKII activity was unaffected by a 5 min exposure to NO, it was significantly reduced after prolonged exposure times (Fig.  3B). A similar time-dependent reduction was also observed for FIGURE 2. NO induces Ca 2؉ -independent autonomous CaMKII activity by a process that requires both Cys-280 and Cys-289. A, incubation of CaMKII (in the presence of Ca 2ϩ /CaM) either with the oxidizer H 2 O 2 or the NO donor DEA-NONOate for 5 min generated autonomous CaMKII activity (measured by phosphorylation of syntide 2 after chelating Ca 2ϩ with EGTA). NO-induced autonomy was abolished by mutation of either Cys-280 or Cys-289. Bar graphs indicate absolute kinase activity as mean Ϯ S.E. The numbers inserted for each bar indicate the level of autonomy (in percent of maximal Ca 2ϩ /CaMstimulated activity). B, NO release from the NO donor was readily triggered in buffer at pH 7.2, but not in acidic deionized water. C, NO-induced CaMKII autonomy (measured as in panel A) was not reduced by T286A mutation or by the Ca 2ϩ /CaM competitive inhibitor KN93 (10 M), but effectively blocked by the peptide inhibitor tatCN21 (5 M). These experiments were done with CaMKII from crude HEK cell extracts, and non-transfected mock extracts were thus used as additional control. Bar graphs indicate mean Ϯ S.E. the NO-induced autonomous activity (Fig. 3B), indicating that the observed reduction of CaMKII activity was not mediated by interference with Ca 2ϩ /CaM-stimulation. Thus, while NO can indeed inhibit both stimulated and autonomous CaMKII activity, this opposing effect requires prolonged NO signaling, while shorter NO signals are sufficient to generate autonomous activity.
NO Reduces Ca 2ϩ /CaM-induced CaMKII Thr-286 Autophosphorylation-For other forms of Thr-286-independent forms of CaMKII autonomy, a positive cross regulation with Thr-286induced autonomy has been suggested (10 -12). By contrast, previous exposure to NO in presence of Ca 2ϩ /CaM dramatically reduced subsequent CaMKII autophosphorylation at Thr-286, which was induced by addition of ATP (Fig. 4A). While both Thr-286 phosphorylation and NO generate autonomous CaMKII activity, the level of Thr-286-induced autonomy is higher: While autonomy by Thr-286 phosphorylation is ϳ20% of maximally stimulated activity (8,16), autonomy by NO was only ϳ10% (see Fig. 2A). Indeed, addition of NO donors prior to a Thr-286 autophosphorylation reaction reduced not only Thr-286 phosphorylation (Fig. 4A), but also the resulting autono-mous activity (Fig. 4B). A similar effect was caused by the oxidizer H 2 O 2 (Fig. 4B) that generated a lower level of autonomy than NO (see Fig. 2A).
This finding raised the possibility that generation of autonomous CaMKII activity by NO exposure may even substitute for Thr-286 phosphorylation in enabling efficient Thr-305/306 phosphorylation. However, NO exposure did not cause any significant increase in Thr-305 autophosphorylation for CaMKII that was not previously Thr-286 phosphorylated (Fig. 5).
NO-induced CaMKII Autonomy Is Mediated by Specific S-Nitrosylation-In addition to S-nitrosylation of cysteine residues, NO can induce oxidation of cysteine and methionine residues, the latter through formation of peroxynitrite (ONOO Ϫ ). However, four independent lines of evidence showed that the NO-induced CaMKII autonomy was indeed caused by S-nitrosylation of Cys-280/Cys-289 and not by ONOO Ϫ -mediated oxidation: (i) The effect of NO donors was larger compared with oxidation by H 2 O 2 (see Fig. 2A), consistent with a bulkier modifica-   JULY 11, 2014 • VOLUME 289 • NUMBER 28

JOURNAL OF BIOLOGICAL CHEMISTRY 19461
tion by nitrosylation causing more efficient relief from autoinhibition.
(iii) The ONOO Ϫ scavengers tryptophan (Trp) or MnTMPyP (MnTP) did not reduce the effect of NO donors, but instead even further enhanced it (Fig. 7A), consistent with suppression of Cys-280/289 oxidation allowing more nitrosylation of these residues. Comparing scavenger addition before and after the nitrosylation reaction (both before the kinase activity assays) confirmed that the scavengers affected the nitrosylation reaction and did not have any other direct effects on kinase activity (Fig. 7A).
(iv) The effect of directly adding ONOO Ϫ was much smaller compared with adding NO donors. Importantly, the effect of ONOO Ϫ was abolished by the ONOO Ϫ scavenger Trp, but dramatically enhanced by MnTP, an ONOO Ϫ scavenger that releases NO during ONOO Ϫ consumption (Fig. 7B).

NO-induced Cell Death Is Reduced by CaMKII Inhibition-
In dissociated hippocampal neuron cultures, similar levels of cell death were triggered by excitotoxic glutamate and by direct application of NO donors (Fig. 8). Importantly, CaMKII inhibition with tatCN21 significantly protected from cell death not only when induced by glutamate (6, 7), but also when induced by NO donors (Fig. 8). Notably, both glutamate-and NO-induced cell death were also reduced by inhibiting the NMDAtype glutamate receptor with APV (Fig. 8), indicating that NOinduced cell death also required Ca 2ϩ influx through this receptor (as induced by the high spontaneous neuronal activity within the cultures). The observed Ca 2ϩ requirement for NOinduced cell death is also consistent with the regulation of NOinduced CaMKII autonomy, which also required a Ca 2ϩ stimulus (see Fig. 3A).

DISCUSSION
The results of this study demonstrate that NO-induced CaMKII autonomy is a novel regulatory mechanism for an enzyme critically involved in mediating synaptic plasticity (1,(3)(4)(5) and ischemic/excitotoxic neuronal cell death (6,7). Indeed, CaMKII inhibition protected also from neuronal cell death directly induced by NO donors. NO can cause S-nitrosylation or oxidation of proteins (the latter via formation of ONOO Ϫ ); however, the results of this study showed that CaMKII autonomy was induced by specific S-nitrosylation that occurs at residues Cys-280/289. Mutation of either residue abolished NO-induced autonomy, indicating requirement of their simultaneous nitrosylation.
Generation of CaMKII autonomy by autophosphorylation at Thr-286 has been considered to be a hallmark feature of CaMKII regulation for almost 30 years, as it enables the kinase to "remember" past Ca 2ϩ stimuli (8, 14 -16) (for review see Ref. 1). Several additional ways to generate autonomous CaMKII activity have been prominently described more recently: Binding to GluN2B (10), Met-280/281 oxidation (11), and Ser-279 O-linked glucosylation (12). The novel form of autonomy that is generated by NO-induced S-nitrosylation shares several features with these previously described autonomy mechanisms. All known forms of CaMKII autonomy require an initial Ca 2ϩ / CaM-stimulus for induction (8 -12). Thus, they all provide a molecular memory of past Ca 2ϩ stimuli. However, in case of autonomy induced by oxidation, glucosylation, or S-nitrosylation, this molecular memory is only formed in presence of a coinciding second type of signal. For oxidation and glucosylation, the second signal appears to be typically pathological (such as oxidative stress or elevated blood sugar in diabetes; Refs. 11,12). By contrast, NO signals that are required for S-nitrosylation could additionally be generated by physiological signaling. All forms of autonomous CaMKII are also significantly further stimulated by Ca 2ϩ /CaM (for review see Ref. 1). This can retain Ca 2ϩ sensitivity even of "autonomous" CaMKII and may prevent complete uncoupling from subsequent Ca 2ϩ stimuli. Indeed, recent evidence sug-  gests that autonomous CaMKII activity can have opposing effects on synaptic strength, depending on presence or absence of additional Ca 2ϩ /CaM stimulation (5).
With ϳ20% of maximal Ca 2ϩ /CaM-stimulated activity, Thr-286 phosphorylation likely generates the highest level of CaMKII. While S-nitrosylation generated lower levels of autonomy (ϳ10 -15%), this was significantly higher than autonomy induced by oxidation (ϳ5%), an effect that may be correlated to the bulk of the respective modification, as well as with the position of the modified residues within the auto-inhibitory region of the regulatory domain (see Fig. 1). Autonomy induced by NO is likely also higher compared with autonomy induced by GluN2B binding or glycosylation, although this is more difficult to compare. Assessment of GluN2B induced autonomy (10,25) was done using a substrate peptide that enhances autonomy through a special mechanism not shared with normal substrates (16). Consequently, the actual level of autonomy is likely lower than originally reported (10,25) and ϳ5-10% autonomy was put forward as a more likely estimate (1). The level of autonomy induced by glucosylation (specifically O-linked N-acetylglucosamine modification of Ser-279) is unclear (12). Using the Camui FRET-sensor of CaMKII activity (33), conditions that induced CaMKII glucosylation caused the same extent of loss in FRET as full stimulation with Ca 2ϩ /CaM (12). However, these conditions increased actual CaMKII activity only 1.7-fold over baseline, and activity in response to full Ca 2ϩ / CaM stimulation was not compared (12). Based on the ϳ1000fold stimulation of CaMKII by Ca 2ϩ /CaM (1), these results could indicate as little as 0.17% autonomy (or up to 7% autonomy, in case that the baseline was largely due to background and contribution of basal CaMKII activity was only 1%). Furthermore, while the FRET change was clearly sensitive to mutation of the glucosylated residue, this remains to be tested for the modest increase in actual CaMKII activity (12). Also, while specific CaMKII glucosylation clearly had important functional consequences, it is still unclear if these function were mediated by autonomous CaMKII activity, by stimulated activity, or by activity-independent CaMKII effects (12), especially since examples of the latter have been described for some neuronal CaMKII functions (34,35). Obviously, the FRET sensor reports structural changes rather than actual kinase activity, and the sensor is known to over-report Thr-286-dependent autonomous activity (12,33). Notably, this over-reporting can be an advantage for the detection of autonomous CaMKII compared with the significantly larger activity of stimulated CaMKII (36). However, it should also be noted that there is a novel FRET sensor of CaMKII activity (with different placement of the fluorescent proteins) that more accurately reports the levels of autonomous activity relative to stimulated levels, at least for the Thr-286-dependent autonomy (37).
The secondary effects of the different forms of CaMKII autonomy on levels of Ca 2ϩ /CaM-stimulated activity also differ. Thr-286 phosphorylation mildly enhances stimulated activity by 10 -20% (26), but also promotes additional Thr-305/ 306 phosphorylation that prevents subsequent Ca 2ϩ /CaM binding (26,31,32,38). By contrast, GluN2B binding actually reduces stimulated activity (10,39), but inhibits Thr-305/306 phosphorylation (10) (for review see Ref. 1). By contrast S-nitrosylation did not directly affect Ca 2ϩ /CaM-stimulated activity, but inhibited subsequent Thr-286 phosphorylation (with no effects on Thr-305 phosphorylation). This prevented genera- FIGURE 7. NO induces CaMKII autonomy via S-nitrosylation. A, ONOO Ϫ scavengers tryptophan (Trp) and MnTMPyP (MnTP) did not block but enhance the NO donor effect on CaMKII autonomy. The scavengers had no effect on CaMKII activity when instead added only immediately before the kinase assays. B, direct ONOO Ϫ addition resulted only in a small increase in CaMKII autonomy, which was blocked by Trp but enhanced by MnTP, an ONOO Ϫ scavenger that releases NO during ONOO Ϫ consumption. The quantifications indicate mean Ϯ S.E. tion of the higher level of Thr-286-dependent autonomy, an effect observed here also after CaMKII oxidation. Thus, the different forms of CaMKII autonomy can have intricate crossregulatory effects with each other as well as with stimulated CaMKII activity. NO-induced CaMKII autonomy is a novel aspect also in the cross-talk between CaMKII and different NOS isoforms, as these NO-producing enzymes are in turn regulated by CaMKII activity (40 -45).
Both CaMKII and neuronal NOS (nNOS) are localized at synapses via association with the NMDA-receptor complex, are involved in excitotoxic/ischemic cell death, and inhibition of either is neuroprotective (for review see Refs. 7, 23). The CaMKII-mediated regulation of nNOS cannot explain this effect, as CaMKII inhibits rather than activates nNOS (40,43,44). By contrast, the NO-induced CaMKII autonomy indicates that nNOS is instead upstream of CaMKII in excitotoxic death signaling. Indeed, CaMKII inhibition protected not only from excitotoxic/ischemic death, but also from neuronal cell death directly induced by an NO donor. Intriguingly, even disruption of nNOS from the NMDA-receptor complex provides neuroprotection (22), and it will be interesting to see if this disruption acts by preventing efficient S-nitrosylation of the co-localized CaMKII.
Two recent studies indicated NO-mediated regulation of CaMKII also in the heart (46,47), where CaMKII is known to have crucial pathological functions (13). However, in this case, CaMKII activation by NO did not appear to be through direct S-nitrosylation, as CaMKII activation (and the downstream functions) was instead dependent on PKG (47), which is activated by cGMP after NO-induced guanylyl cyclase activation (17)(18)(19). Notably, however, one of these studies showed that NO donors did also cause a direct 2.5-fold increase of CaMKIIó activity over baseline (46). However, this rather small effect may have been caused by ONOO Ϫ -mediated oxidation (an effect that was ruled out in our experiments on the CaMKII␣ isoform). Indeed, in this case, the NO effect was no larger than the effect of oxidation (46), and the CaMKIIó isoform does not have a Cys residue at the position homologous to Cys-280 in CaMKII␣ (and the nitrosylation effect on CaMKII␣ autonomy demonstrated here was sensitive not only to mutation of the Cys-289 that is conserved among the isoforms, but also to mutation of the unique Cys-280).
It should be noted that the absolute activity of CaMKII made autonomous by oxidation reported here (ϳ1 mol/min/mg) is higher than previously reported, while the level of relative autonomy (ϳ5% of maximal stimulated activity) is lower than reported previously (ϳ25 nmol/min/mg and ϳ40%, respectively) (11). The previously reported relative autonomy may be an overestimate due to kinase reactions that were beyond the linear range, for instance due to long reaction times (1). This is consistent with the low apparent activity reported after maximal stimulation and with the high apparent autonomy also after Thr-286 phosphorylation (70%) (11), even though the assays used a regular substrate described to support only lower levels of ϳ20% autonomy (8,16). However, as previously discussed (1), we wish to point out that such overestimates of the relative autonomy levels after oxidation (11) or GluN2B binding (10) do not diminish the importance of these studies that elucidated new principle pathways toward CaMKII autonomy. The NO mechanisms described here add another path to CaMKII autonomy, and it will be interesting to investigate the functions in physiological CaMKII signaling, in addition to the pathological functions in neuronal cell death described here.