Purification and characterization of calmodulin-dependent protein kinase II from rat spleen: a new type of calmodulin-dependent protein kinase II

A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 A, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, calmodulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logarithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50,000 to 60,000 which can bind to calmodulin. Thus a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.     

Autoactivation of calmodulin-dependent protein kinase II by autophosphorylation

Autophosphorylation of calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) under limiting conditions (2 microM ATP) decreased progressively with increasing concentrations of a substrate, Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (syntide-2), suggesting a competition between the substrate and the autophosphorylation site(s) of the enzyme. The rate and extent of the generation of Ca2+/CaM-independent activity of the enzyme by autophosphorylation were also decreased by the presence of syntide-2. The syntide-2 phosphorylation in the presence of Ca2+/CaM under the limiting conditions reached a steady state, after a lag, when the Ca2+/CaM-independent activity reached a plateau. A linear relationship was observed between the activities in the presence and absence of Ca2+/CaM of the enzyme which had undergone various degrees of autophosphorylation, and the extrapolation of activity in the absence of Ca2+/CaM to zero gave 15-20% of the maximum activity. The steady-state rate of syntide-2 phosphorylation in the presence of Ca2+/CaM by the enzyme that had not undergone prior autophosphorylation was decreased by high concentrations of syntide-2 which suppressed autophosphorylation as well as the generation of Ca2+/CaM-independent activity. These results suggest that although the nonautophosphorylated enzyme possesses a basal low level of Ca2+/CaM-dependent activity, autophosphorylation is required for full activation.     

Calcium . calmodulin-dependent protein kinase II and calcium . phospholipid-dependent protein kinase activities in rat tissues assayed with a synthetic peptide

Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases.     

Calcium/calmodulin-dependent protein kinase II and synaptic plasticity

A prominent role for calcium/calmodulin-dependent protein kinase II (CaMKII) in regulation of excitatory synaptic transmission was proposed two decades ago when it was identified as a major postsynaptic density protein. Since then, fascinating mechanisms optimized to fine-tune the magnitude and locations of CaMKII activity have been revealed. The importance of CaMKII activity and autophosphorylation to synaptic plasticity in vitro, and to a variety of learning and memory paradigms in vivo has been demonstrated. Recent progress brings us closer to understanding the regulation of dendritic CaMKII activity, localization, and expression, and its role in modulating synaptic transmission and cell morphology.     

Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II

Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin.     

Further comparison of calmodulin-dependent protein kinase II from brain and calmodulin-dependent glycogen synthase kinase from skeletal muscle

Calmodulin-dependent protein kinase II was purified from rabbit brain and its properties were compared with those of calmodulin-dependent protein kinase II from rat brain and calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Rabbit brain calmodulin-dependent protein kinase II was clearly distinguished from rabbit skeletal muscle glycogen synthase kinase with respect to size, behavior on autophosphorylation, immunological cross-reactivity and peptide mapping, but was indistinguishable from rat brain calmodulin-dependent protein kinase II in all respects examined. Thus, differences between calmodulin-dependent protein kinase II and glycogen synthase kinase appear not to reflect a species difference but to reflect a tissue difference.     

Self-regulation of calmodulin-dependent protein kinase II and glycogen synthase kinase by autophosphorylation

Calmodulin-dependent protein kinase II from rat brain underwent autophosphorylation and the autophosphorylation caused a marked decrease in the enzyme activity. Calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle was also inactivated by incubation under autophosphorylating conditions. The inactivation of the protein kinases by the autophosphorylation may be an important self-regulatory mechanism in controlling the enzyme activities.     

Phosphorylation and activation of Ca2+/calmodulin-dependent protein kinase phosphatase by Ca2+/calmodulin-dependent protein kinase II.

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKPase) is a protein phosphatase which dephosphorylates autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) and deactivates the enzyme (Ishida, A., Kameshita, I. and Fujisawa, H. (1998) J. Biol. Chem. 273, 1904-1910). In this study, a phosphorylation-dephosphorylation relationship between CaMKII and CaMKPase was examined. CaMKPase was not significantly phosphorylated by CaMKII under the standard phosphorylation conditions but was phosphorylated in the presence of poly-L-lysine, which is a potent activator of CaMKPase. The maximal extent of the phosphorylation was about 1 mol of phosphate per mol of the enzyme and the phosphorylation resulted in an about 2-fold increase in the enzyme activity. Thus, the activity of CaMKPase appears to be regulated through phosphorylation by its target enzyme, CaMKII.     

钙离子／钙调素依赖性蛋白激酶Ⅱ及其功能

所有引起细胞内钙离子浓度升高的激素或神经递质都可通过不同的钙离子／钙调素依赖性蛋白激酶达到调节细胞生理功能的作用。在神经元活动、细胞分泌、平滑肌缩等 细胞活动中起重要作用。     

Conformational changes underlying calcium/calmodulin-dependent protein kinase II activation

Calcium/calmodulin-dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.     

Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.     

Selection of peptide inhibitors for double-stranded RNA-dependent protein kinase PKR

Protein kinase inhibitors have been developed and applied as antitumor drugs. The majority of these inhibitors are derived from ATP analogs with limited specificity towards the kinase target. Here we present our proof-of-principle study on peptide inhibitors for kinases. Two peptides were selected by phage display against double-stranded RNA-dependent protein kinase (PKR). In vitro assay revealed that these peptides exhibit an inhibitory effect on PKR-catalyzed phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). The peptides also interrupt PKR activity in cells infected by viruses, as PKR activation is one of the hallmarks of host response to viral infection. Kinetic study revealed that one of the peptides, named P1, is a competitive inhibitor for PKR, while the other, named P2, exhibits a more complicated pattern of inhibition on PKR activity. Fragment-based docking of the PKR-peptide complex suggests that P1 occupies the substrate pocket of PKR and thus inhibits the binding between PKR and eIF2α, whereas P2 sits near the substrate pocket. The computational model of PKR-peptide complex agrees with their kinetic behavior. We surmise that peptide inhibitors for kinases have higher specificity than ATP analogs, and that they provide promising leads for the optimization of kinase inhibitors.     

Ca2+/calmodulin-dependent protein kinase II is required for microcystin-induced apoptosis.

The potent natural toxins microcystin, nodularin, and okadaic acid act rapidly to induce apoptotic cell death. Here we show that the apoptosis correlates with protein phosphorylation events and can be blocked by protein kinase inhibitors directed against the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The inhibitors used comprised a battery of cell-permeable protein kinase antagonists and CaMKII-directed peptide inhibitors introduced by microinjection or enforced expression. Furthermore, apoptosis could be induced by enforced expression of active forms of CaMKII but not with inactive CaMKII. It is concluded that the apoptogenic toxins, presumably through their known ability to inhibit serine/threonine protein phosphatases, can cause CaMKII-dependent phosphorylation events leading to cell death.     

Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II

Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) isozyme variability is the result of alternative usage of variable domain sequences. Isozyme expression is cell type-specific to transduce the appropriate Ca(2+) signals. We have determined the subcellular targeting domain of delta(E) CaMK-II, an isozyme that induces neurite outgrowth, and of a structurally similar isozyme, gamma(C) CaMK-II, which does not induce neurite outgrowth. delta(E) CaMK-II co-localizes with filamentous actin in the perinuclear region and in cellular extensions. In contrast, gamma(C) CaMK-II is uniformly cytosolic. Constitutively active delta(E) CaMK-II induces F-actin-rich extensions, thereby supporting a functional role for its localization. C-terminal constructs, which lack central variable domain sequences, can oligomerize and localize like full-length delta(E) and gamma(C) CaMK-II. Central variable domains themselves are monomeric and have no targeting capability. The C-terminal 95 residues of delta CaMK-II also has no targeting capability but can efficiently oligomerize. These findings define a targeting domain for gamma and delta CaMK-IIs that is in between the central variable and association domains. This domain is responsible for the subcellular targeting differences between gamma and delta CaMK-IIs.