Binding of CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr(286)-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S(0.5) (substrate concentration at half maximal velocity) and V(max) values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B((1271-1311)) (with the phosphorylation site Ser(1303)) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST-NR2A was significantly altered by the interaction with GST-NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of alpha-CaMKII, Glu(96), participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr(286)-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

Ca2+ influx through NMDA‐type glutamate receptor at excitatory synapses causes activation of post‐synaptic Ca2+/calmodulin‐dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B‐Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca2+/calmodulin activated form and the autonomously active Thr286‐autophosphorylated form of CaMKII. Green fluorescent protein–α‐CaMKII co‐expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione‐S‐transferase pull‐down assay. Thr286‐autophosphorylated α‐CaMKII or the autophosphorylation mimicking mutant, T286D‐α‐CaMKII, binds NR2B sequence independent of Ca2+/calmodulin unlike native wild‐type α‐CaMKII. We show enhancement of this binding by Ca2+/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B‐S1303D) abolishes the Ca2+/calmodulin‐independent binding whereas it allows the Ca2+/calmodulin‐dependent binding of α‐CaMKII in vitro. Similarly, the autonomously active mutants, T286D‐α‐CaMKII and F293E/N294D‐α‐CaMKII, exhibited Ca2+‐independent binding to non‐phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho‐Ser1303 on NR2B.

Advances in proteome research have opened the gateway to understanding numerous metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. In the present study, the antioxidant capacity of four tomato genotypes i.e., Kashi Amrit, Kashi Anupam, EC-317-6-1, and WIR-4360 was determined under drought stress to ascertain the scavenging potential for reactive oxygen species (ROS). A significant increase in the superoxide dismutase (SOD), Ascorbate peroxidase (APX), and catalase (CAT) activities in all the four genotypes under drought stress was observed, which seemed to be associated with a protective role against ROS (p < 0.001). Based on the antioxidant enzyme activities, a proteomic approach was applied to study differential protein expression in two selected genotypes from different species i.e., EC-317-6-1 (Solanum pimpinellifolium) and Kashi Amrit (Solanum lycopersicum) grown under irrigated, drought, and re-watering conditions. To reveal the protein network regulated under these conditions, two-dimensional gel electrophoresis was employed to identify and quantify the number of proteins in drought-sensitive (Kashi Amrit) and tolerant (EC-317-6-1) genotypes. Matrix-assisted laser desorption/ionization-time of flight analysis (MALDI-TOF) revealed a total of 453 spots after fine-tuning factors i.e., smoothness, saliency, and minimum area that responded to drought. Out of 453 total spots, 93 spots were identified in Kashi Amrit and 154 in EC-317-6-1 under irrigated conditions, whereas 4 spots were identified in Kashi Amrit and 77 spots in EC-317-6-1 under drought conditions. Furthermore, differentially expressed proteins were distinguished according to the fold change of their expression. Information provided in this report will be useful for the selection of proteins or genes in analyzing or improving drought tolerance in tomato cultivars. These findings may assist in the construction of a complete proteome database encompassing various divergent species which could be a valuable source for the improvement of crops under drought-stress conditions in the future.

CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation.

Abiotic stresses are one of the key limitations to global crop production and food security. Among the abiotic stresses, drought is one of the most vital factors that causes change in morphological, biochemical and physiological characteristics in plants, and consequently affects the growth and productivity of crops. The main purpose of the present study was to evaluate the effect of drought on morphological [Plant height, root length (cm), shoot length (cm), number of branches, yield attributing traits], physiological ratio of root/shoot length, leaf area (cm 2 ), relative water content (%), and electrolyte leakage (% conductivity) and biochemical traits [ascorbic acid content (mg/100g), total carotenoids (mg/100g), total chlorophyll content, proline, sugar content] in 15 tomato genotypes and to identify drought stress tolerant genotypes. The results confirmed that there are significant variations in agronomic, physiological and biochemical parameters among 15 tomato genotypes under drought and irrigated conditions. Among the 15 genotypes, EC-317-6-1 and WIR-4360 were found highly tolerant to drought in comparison to others while Kashi Amrit and Kashi Sharad were found susceptible to drought conditions. The performance of tomato genotypes used in the study showed significant differences in all studied traits, suggesting that they could be taken into account when selecting for drought tolerance. EC-317-6-171 and WIR-4360 had good yield performance under deficit irrigation treatment. Moreover, results indicate that biochemical and physiological parameters are more useful for the screening of drought tolerant tomato genotypes.