A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity

Radiation Induces Acute Alterations in Neuronal Function

Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage. In the past it was accepted that radiation-induced normal tissue injury resulted from a progressive reduction in the survival of clonogenic cells. Moreover, because radiation-induced brain dysfunction is believed to evolve over months to years, most studies have focused on late changes in brain parenchyma. However, clinically, acute changes in cognition are also observed. Because neurons are fully differentiated post-mitotic cells, little information exists on the acute effects of radiation on synaptic function. The purpose of our study was to assess the potential acute effects of radiation on neuronal function utilizing ex vivo hippocampal brain slices. The cellular localization and functional status of excitatory and inhibitory neurotransmitter receptors was identified by immunoblotting. Electrophysiological recordings were obtained both for populations of neuronal cells and individual neurons. In the dentate gyrus region of isolated ex vivo slices, radiation led to early decreases in tyrosine phosphorylation and removal of excitatory N-methyl-D-aspartate receptors (NMDARs) from the cell surface while simultaneously increasing the surface expression of inhibitory gamma-aminobutyric acid receptors (GABAARs). These alterations in cellular localization corresponded with altered synaptic responses and inhibition of long-term potentiation. The non-competitive NMDAR antagonist memantine blocked these radiation-induced alterations in cellular distribution. These findings demonstrate acute effects of radiation on neuronal cells within isolated brain slices and open new avenues for study

Improving a Natural CaMKII Inhibitor by Random and Rational Design

CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction

The CaMKII Holoenzyme Structure in Activation Competent Conformations

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ~20% appear to form dimers an

CaMKII T305 phosphorylation is further reduced for the T286A mutant.

<p><i>A</i>, T305 phosphorylation was detected by Western analysis after reactions corresponding to the kinase activity assays shown in Fig. 1 (1 min at 30°C, but without substrate peptide) or after extended reaction times (10 min). <i>B</i>, Quantification of the relative T305 phosphorylation by the ratio of the phospho-T305 and total CaMKII immuno-detection values (IDV) shows significantly lower phosphorylation of the T286A mutant in our 1 min kinase assay conditions (N = 3 different extracts for each kinase form; *: p<0.05 in two-tailed t-test). The IDV ratio for T286A was ∼28% (±7.6%) of wild type after the 1 min reactions; in the 10 min reaction experiment shown in panel A, this ratio was ∼83%, consistent with the faster wild type reaction reaching saturation faster.</p

CaMKII T305/306 “burst” auto-phosphorylation <i>in vitro</i>.

<p><i>A</i>, The sequence of the CaMKII regulatory domain with T286 in the autoinhibitory region and T305/306 in the CaM-binding region indicated. <i>B</i>, T305/306 phosphorylation was assessed by Western analysis. CaMKII was pre-phosphorylated at T286 on ice; the “burst” was induced by EGTA addition at 30°C or on ice, and stopped after different reaction times. Note the band-shift caused by phosphorylation of additional sites during the “burst” at 30°C, and the basal immuno-detection without phosphorylation reaction. <i>C</i>, Quantification of the relative T305/306 phosphorylation during the “burst” by arbitrary relative immuno-detection values (IDV). Basal immuno-detection prior to the phosphorylation reactions was subtracted in the quantification shown. No increase in phosphorylation was detected on ice. For 30°C, the results indicate an initial phosphorylation rate (at reaction times of 1 min or less) of ∼0.45 min⁻¹ (assuming that saturation represents near-complete phosphorylation; otherwise the rate is even lower). <i>D</i>, The basal immuno-detection with the phospho-T305 antibody prior to the phosphorylation reactions was likely due to background immuno-reactivity, as indicated by comparison to T305/306A mutant CaMKII.</p

Submaximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 0.1 µM Ca²⁺/CaM and measured by the phosphorylation rate of the substrate syntide 2 (in 1 min reactions at 30°C).

<p>Error bars indicate mean ± s.e.m; **: p<0.01, n.s.: p>0.05 in two-tailed t-test. <i>A</i>, T286 autophosphorylation of CaMKII wild type was assessed by Western analysis (left), and quantified by arbitrary relative immuno-detection values (IDV; right). T286 autophosphorylation stimulated by 0.1 µM Ca²⁺/CaM was slower compared to stimulation by 1 µM Ca²⁺/CaM, but the same level of maximal autophosphorylation was still achieved within 1 min reaction time at 30°C. <i>B</i>, Submaximal activation by 0.1 µM Ca²⁺/CaM was verified by comparing one individual preparation of each CaMKII wild type and T286A mutant to the activity induced by 1 µM Ca²⁺/CaM (n = 4 individual assays). <i>C</i>, The average activity of CaMKII from multiple preparations (N = 5) stimulated by 0.1 µM Ca²⁺/CaM did not differ significantly between CaMKII wild type and the T286A mutant. While the ratio of the mean activities of T286A over wild type was similar as observed at maximal stimulation (compare Fig. 1B,C), the variability at submaximal stimulation was greater (with standard deviations of 35–36% of the mean at submaximal stimulation compared to 13–17% at maximal stimulation for syntide 2 substrate).</p