Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates

Abstract

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.

Members of the plant NIMA‐related kinases are involved in organ development and vascularization in poplar, Arabidopsis and rice

“…This region is specifically targeted by several protein kinase inhibitors (Booher et al. , 1997; Cheetham, 2004; Taylor and Radzio‐Andzelm, 1997), and single amino acid mutations in its sequence have been shown to alter its protein kinase substrate affinity and activity (Praseeda et al. , 2004; Yamaji et al.…”

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin‐dependent protein kinase II

“…The cDNA of α‐CaMKII was amplified by PCR from pGEM2α (Praseeda et al. 2004a) using suitable primers to yield a PCR product with Kpn I and Bam HI sites at 5′ and 3′ ends respectively.…”

“…Site‐directed mutagenesis was carried out using the QuikChange site‐directed mutagenesis Kit based on Kunkel’s method (Kunkel 1985). The cDNA of α‐CaMKII present in the expression vectors pEGFP‐C1‐α‐CaMKII or pFastBac1‐α‐CaMKII (Praseeda et al. 2004a) that was used for expression of the wild‐type (WT) enzyme served as the template to generate mutants.…”

“…The crude insect cell lysate expressing α‐CaMKII and purified α‐CaMKII were prepared as described before (Praseeda et al. 2004a) with modifications.…”

Protection of α-CaMKII from Dephosphorylation by GluN2B Subunit of NMDA Receptor Is Abolished by Mutation of Glu96 or His282 of α-CaMKII

“…WT-α-CaMKII and E96A-α-CaMKII mutant were expressed in Sf21 or High Five insect cells. The crude insect cell lysate and the purified enzymes were prepared as described earlier [ 27 , 37 , 38 ].…”