Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.

CaMKII2 plays a ubiquitous and central role in calcium signaling. Alternative splicing of the four CaMKII genes (␣, ␤, ␥, and ␦) gives rise to ϳ30 known mRNA/protein products. CaMKII␣ and CaMKII␤ predominate in the brain and are involved in normal regulation of synaptic transmission (1-4). Calcium/calmodulin-dependent autophosphorylation at Thr 286 (numbered as in CaMKII␣) enhances the affinity for calcium/calmodulin and confers autonomous (calcium-independent) activity after calmodulin dissociates. Consequently, CaMKII is capable of integrating information conveyed by the amplitude, frequency, and duration of local calcium transients to which it is exposed. Autophosphorylation at Thr 305/306 occurs only in the absence of bound calcium/calmodulin and blocks subsequent calmodulin binding (reviewed in Ref. 5).Knock-in mutation of the Thr 286 or Thr 305/306 autophosphorylation sites in murine CaMKII␣ to Ala or Asp drastically alters some forms of hippocampal synaptic plasticity and disrupts spatial learning behaviors (reviewed in Ref. 6). The specificity of changes in synaptic responses to their dedicated inputs implies that postsynaptic actions of CaMKII, such as modulation of the trafficking and activity of AMPA-type glutamate receptors, are exquisitely regulated in a spatial and temporal manner (7-9).CaMKII interacts with other proteins that we refer to collectively as CaMKII-associated proteins (CaMKAPs). At postsynaptic sites, these include multiple subunits of the N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) (10 -13), densin-180 (14, 15), ␣-actinin (15, 16), cyclin-dependent protein kinase 5 (16), synGAP␤ (17), and filamentous actin (18,19). However, specific contributions of each of these interactions to neuronal signaling and the regulation of synaptic plasticity remain unclear (reviewed in Refs. 7-9). To specifically address this issue, it is critical to have a thorough understanding of the molecular bases for these interactions and the factors regulating complex formation.Prior studies have identified dissimilar high affinity CaMKII-binding domains in the NR2B subunit of the NMDA receptor and in densin-180. These interactions are differentially regulated by calcium/calmodulinbinding, autophosphorylation at Thr 286 , and phosphorylation of the binding proteins (10, 11, 13-15, 20, 21). Moreover, densin-180 and NR2B do not compete with each other for binding to CaMKII (14). Interestingly, densin-180 makes an additional direct interaction with ␣-actinin (15), and ␣-actinin can bind to the NR1 and NR2B subunits of the NMDA receptor (21-24). However, little is known about the molecular determinants for CaMKII binding to ␣-actinin. Here, we report an initial molecular dissection of the CaMKII-binding domain in ␣-actinin-2 and explore the relationship of this interaction to CaMKII binding with densin-180 and NR2B. The data indicate that CaMKII itself may serve as a structural scaffold for the assembly of a postsynaptic signalosome. MATERIALS AND METHODSAntibodies-Western blotting was performed wit...

Postsynaptic roles of CaMKII␣ in synaptic plasticity are thought to require specific targeting to the postsynaptic density (PSD), a cytoskeletal structure juxtaposed to excitatory synapses that is enriched in neurotransmitter receptors and other signaling proteins. Immunohistochemical studies have revealed a wide range in the amount of CaMKII associated with individual PSDs (4, 5), consistent with a dynamic regulation of PSD targeting. Exogenous, Thr 286 -autophosphorylated CaMKII␣ ([P-T 286 ]CaMKII) binds specifically to isolated PSDs (6). Moreover, pharmacological treatments of hippocampal slices that activate CaMKII and induce long term potentiation, a form of synaptic plasticity, increase the amount of CaMKII associated with PSD-enriched subcellular fractions (6). Elegant studies of green fluorescent protein-CaMKII localization in cultured hippocampal neurons suggest that transient translocation of soluble CaMKII␣ to synapses in response to NMDA-type glutamate receptor (NMDAR) activation requires only Ca 2ϩ /calmodulin binding to CaMKII (7). However, mutagenesis studies indicate that Thr 286 autophosphorylation stabilizes the synaptic targeting of green fluorescent protein-CaMKII␣, whereas Thr 305/306 autophosphorylation promotes dissociation of synaptic green fluorescent protein-CaMKII␣ (8). Consistent with these data, the transgenic mouse knock-in mutation of Thr 305 in CaMKII␣ to Asp shows reduced CaMKII association with the PSD and changes in long term potentiation and learning and memory (2, 9, 10). Interestingly, a mouse model of Angelman mental retardation syndrome that has deficits in long term potentiation and spatial learning also displays decreased PSD-associated CaMKII␣ and increased autophosphorylation at Thr 305/306 (11). Other studies have shown that PKC activation can also drive synaptic translocation of CaMKII by a mechanism that is dependent on the actin cytoskeleton (12). In combination, these studies suggest that multiple, complex cellular mechanisms control PSD targeting of CaMKII. However, the molecular basis for these dynamic interactions is poorly defined.CaMKII binds several PSD-enriched CaMKII-associated proteins (CaMKAPs), including F-actin (13), cyclin-dependent protein kinase 5 (14), synGAP␤ (15), ␣-actinin (14, 16), densin-180 (16, 17), and multiple NMDAR subunits (18 -21