Catalytic Subunit of PKA as a Prototype of the Eukaryotic Protein Kinase Family.

The catalytic subunit of protein kinase A (PKAc) is conserved in all eukaryotic protein kinases. PKAc consists of two lobes that form the catalytic cleft containing the ATP-binding, peptide-binding site, and catalytic sites. During folding, PKAc secondary structures organize so that the non-polar regions form a globular core, while mobile loops and tails are exposed and can act as regulatory elements. De novo synthesized PKAc is phosphorylated at the T-loop, resulting in the formation of the active center capable of high-affinity binding of co-substrates. The ATP-molecule "sticks" the two lobes together, whereas the binding of peptide substrate completes the active center formation. The resulting catalytic triad (γ-phosphate of ATP, hydroxyl of Ser/Thr residue of the protein substrate, and Asp166 carboxyl) occupies a position optimal for catalysis. During the catalytic cycle, dynamic reorganization of polar and hydrophobic interactions ensures PKAc transition from the open to the closed conformation and vice versa. Understanding the structural basis of functioning of eukaryotic protein kinases (ePKs) is essential for successful design of ePK modulators.

[Effect of etimizole structural analogues on protein kinase CK2, protein phosphorylation and transcription of chromatin in rat brain cortex and hippocampus]. / Vliianie strukturnykh analogov étimizola na proteinkinazu SK2, fosforilirovanie belkov i transkriptsiiu khromatina neironov kory i gippokampa mozga krys.

Protein kinase CK2 is an important enzyme in the nervous system. The nuclear forms of CK2 regulate chromatin structure and gene expression, the key processes for long-term memory formation. Memory modulators, the Structural Analogues of Etimizole (SAE), were able to increase or decrease the activity of chromatin-associated CK in the cortex and hippocampus of rat brain in vitro. In vivo memory enhancers from SAE-group (3 mg/kg) stimulated CK2 activity and the transcriptional ability of chromatin in the cortex and hippocampus, starting from 30 min with a peak for 60 min and a duration up to 180 min. At these periods the memory inhibitor from the SAE-group reduced CK2 activity and chromatin transcription. It is assumed that the modulating effect of SAE on CK2 activity and transcription underlies the effects of these compounds on long-term memory.

[Ethymisole: reassessing and old drug].

Ethymisole, or 4,5-di(N-methylcarbamoyl)-1-ethyl-imidazole, is a cognitive enhancer and nootropic drug, the molecular target of which is a multifunctional protein kinase C K2 (casein kinase II). New data about signal pathways and protein substrates of CK2 have been obtained due to research effort of many laboratories. The paper presents a historical sketch of molecular investigations underlying memory enhancer effects of ethymisole; this and the other pharmacological effects of ethymisole are considered in the light of new data.

Participation of RNA synthesis in the process of the development and maintenance of conditioned reflexes (an investigation using antitheines).

It has been established that an increase in RNA synthesis in the neurons of the cerebral cortex of rats at the stage of consolidation is manifested in well-trained animals more strongly than in poorly trained animals. The selective influence of propylnorantitheine and demethylated derivatives of ethylnorantitheine on the maintenance of conditioned reflexes has been demonstrated. The effects of these substances on consolidation and long-term memory correlate with the change in the RNA-synthesizing activity of neurons during the effect both in systemic experiments and with the direct interaction with the chromatin of the neurons. The participation of the RNA synthesis of cerebral cortical neurons in the mechanisms of long-term memory is discussed.

Involvement of the genetic apparatus in memory formation mechanisms: the role of the neuronal calcium-regulatory system in rats.

Stimulators of long-term memory (ethylnorantiphein and its analogs M1 and M2) were used to study the dynamics of several components of the neuronal calcium-regulatory system in the rat cortex and hippocampus. There were no changes in the activity of the Mg, Ca-ATPase transporter and actomyosin-like Ca-ATPase in synaptosomes 5, 15, 60, and 180 min after dosage with these agents. On exposure to ethylnorantiphein, M1, and M2, activation of RNA transcription at 60 min was accompanied by notable increases in chromatin Ca-ATPase activity, along with an increase in the synthesis of synaptosomal proteins at 180 min, with an increase in synaptic membrane protein kinase C activity. An increase in chromatin Ca-ATPase activity was also seen during fixation of a conditioned active escape reflex. It is suggested that the increase in protein kinase C activity is associated with secondary rearrangements of the synaptic membranes. The question of the role of direct activation of the genetic apparatus by neuroactive substances in the molecular mechanisms of memory formation is discussed.

Status of the "protein kinase CK2-HMG14" system in age-related amnesia in rats.

The experiments described here demonstrate that disruption of the phosphorylation of transcription factors of the HMG cAMP/Ca-independent protein kinase CK2 class may be the cause of decreased gene expression in age-related cognitive deficits. Amnesia for a conditioned passive avoidance reaction (CPAR) in aged rats (24 months old) was accompanied by decreases in the synthesis of synaptosomal proteins and transcription in nuclei isolated from cortical, hippocampal, and striatal neurons. There was a decrease in chromatin protein kinase CK2 activity and a significant decrease in the phosphorylation of HMG14 by protein kinase CK2. Selective activators of protein kinase CK2 (1-ethyl-4-carbamoyl-5-methylcarbamoylimidazole and 1-ethyl-4,5-dicarbamoylimidazole) increased HMG14 phosphorylation by protein kinase CK2, increased transcription, increased the synthesis of synaptosomal proteins, and decreased amnesia for the CPAR in aged rats. Thus, activation of the "protein kinase CK2-HMG14" system is accompanied by optimization of synaptic plasticity in aged animals. The results provide evidence for the high therapeutic potential of protein kinase CK2 activators.

[Therapeutic potential of protein kinase CK2 modulators]. / Terapevticheskii potentsial moduliatorov proteinkinazy CK2.

Data on the nuclear cascade of signal transduction, including protein kinase CK2 (PKCK2), transcription factor HMG14 and chromatin myosin-like proteins, are generalized with regard for the modern understanding of mechanisms of synaptic plasticity. The role of the neurospecific isoform and subunit structure of PKCK2, of the individual subunit autophosphorylation of PKCK2, of phosphorylation of substrate-proteins in the enzyme activity and of conformation transformations of chromatin is examined. Data on changes in the CK2-induced cascade and synaptic plasticity in learning, on age-related amnesia and on cognitive deficits induced by ethanol and chloridine in rat embryos are presented. The prospects for using modulators PKCK2, 4,5-di(N-methylcarbamoyl)-l-alkyl-imidazoles, as potential nootropics are discussed.

[Current aspects of the search of effective nootropic drugs]. / Sovremennye aspekty poiska éffektivnykh nootropnykh sredstv.

The authors review different mechanisms of mnemotropic and cerebroprotective effects of nootropic drugs. The data concerning the molecular mechanisms of action of the structural analogs of the memory stimulant ethylnorantifeine (etimizol) have been summarized and analyzed. It is shown that the effects of antifeines on the retention of the conditioned reflexes are independent of their effects on the cAMP system and structural-functional condition of the neuronal membrane. The key role in the action of these compounds on the long-term memory is played by the activity of the genetic apparatus. The existence of nootropic receptors in neuronal chromatin is assumed.

[The participation of the genetic apparatus in the mechanisms of memory trace formation: the role of the calcium-regulatory system of the neurons in rats]. / Uchastie geneticheskogo apparata v mekhanizmakh sledoobrazovaniia: rol' kal'tsii-reguliatornoi sistemy neironov krys.

The dynamics of changes in some components of the calcium-regulated system of cortical and hippocampal neurons under the influence of long-term memory enhancers (ethylnorantifein and its analogues M1 and M2) was studied in rat brain. No change was found in the activity of transport Mg, Ca-ATPase and actomyosin-like Ca-ATPase in synaptic membranes 5, 15, 60, and 180 min after the injection of memory enhancers. The activation of the RNA transcription (60 min after the injection) was accompanied by an appreciable increase in activity of the chromatin Ca-ATPase. The amplification of synaptosomal protein synthesis (180 min) was accompanied by an increase in the activation of protein kinase C of synaptic membranes. The increase in Ca-ATPase activity of chromatin was also shown during the consolidation of the conditioned active avoidance in rats. The increase in the activity of protein kinase C seems to be connected with secondary rearrangement in synaptic membranes. The role in the long-term memory is discussed of direct activation of the genetic apparatus by neuroactive substances.

[Participation of adenosine receptors in regulating the functional activity of neuronal chromatin by antifeines]. / K voprosu ob uchastii retseptorov adenozina v reguliatsii antifeinami funktsional'noi aktivnosti khromatina neironov.

The regulatory pathways of chromatin transcription in neurons have been studied. The metabolic events (adenylate cyclase, Ca/phospholipid-dependent protein kinase C) involved in signal transduction via adenosine receptors were investigated with the application of pharmacological tools--the memory stimulant ethylnorantifeine and its structural analogs. It has been modelled the biochemical condition of A1 and A2 adenosine receptors in the cortex and the striatum of rat brain. The antifeines did not influence AC of A1 or A2 receptors, unlike the non-metabolizing adenosine derivatives -PIA, NECA and isobutylmethylcanthine. No direct effect of antifeines (10(-7)-10(-3) M) on membrane-bound protein kinase C was established. The data obtained failed to provide evidence for the antifeine effect on the functional activity of chromatin with regard to the triggering role of membrane acceptor systems. The possibility of direct control of the chromatin functional activity by neuroactive drugs is discussed.

Role of autophosphorylation in regulation of protein kinase CK2 from rat neuronal chromatin.

The regulation of rat brain cortex protein kinase CK2 (casein kinase 2) by autophosphorylation has been investigated. Purified CK2 from rat neuronal chromatin is composed of two regulatory (beta) and two catalytic (alpha and/or alpha;) subunits. The molecular masses of the subunits--43 (alpha), 39 (alpha;), and 25 kD (beta)--were similar to those of typical CK2 subunits. A significant amount of alpha;-subunit occurred in neuronal chromatin; the molar ratios for the subunits were 1.1:0.9:1.9. Pharmacological probes (derivatives of 4,5-di(N-methylcarbamoyl)-1-alkyl-imidazole) were used to study autophosphorylation of separate subunits. These compounds have different effects on neuronal chromatin CK2, transcription, and neurological memory. Changes in the autophosphorylation of the CK2 subunits were found with the help of these compounds. Inhibitors of transcription decreased the beta-subunit autophosphorylation. Stimulators of transcription increased the beta-subunit autophosphorylation and promoted the autophosphorylation of the alpha; (but not the alpha) subunit. The beta-subunit autophosphorylation led to reduction in phosphorylation of nonhistone HMG 14 protein that has been shown to be a physiological substrate of CK2. The autophosphorylation of the alpha;-subunit raised the CK2 activity with HMG 14. The question of functional distinctions between the alpha;- and alpha-subunits of chromatin CK2 in differentiated neurons is discussed.

[HMG 14--a physiological substrate for casein kinase NII of neuronal chromatin]. / HMG14--fiziologicheskii substrat kazeinkinazy NII khromatina neironov]

The "in vivo" effects of antifeins on casein kinase NII from rat brain neuronal chromatin have been investigated. Injection of antifeins into rats resulted in changed in cAMP-independent phosphorylation of HMB 14 by casein kinase NII. No changes in HMB 17 phosphorylation were found. Casein kinase NII was isolated and purified from rat brain neuronal chromatin. It was established that casein kinase phosphorylates HMG 14 (but not HMB 17) from rat rain cells and calf thymus as effectively as do exogenous substrates, phosvitin and casein. The Km values for HMB 14 and HMB 17 from brain cells are 5.1 and 180.5 mumol, respectively. Antifeins do not influence casein kinase NII, when HMB 17, phosvitin and casein are used as substrates. HMG 14 phosphorylation changed significantly under the action of antifeins (10(-8)-10(-6) M). "In vivo" and "in vitro", some antifeins increase, and others reduce phosphorylation of HMB 14 by casein kinase NII. This correlates with their action on the transcription and long-term memory. The role of casein kinase NII and its physiological substrates in regulation of chromatin transcription is discussed.

[Participation of protein synthesis in brain cell structures in the action of antifeins on long-term memory]. / Uchastie sinteza belkov tsitostruktur golovnogo mozga v deistvii antifeinov na dolgovremennuiu pamiat'.

Incorporation of 3H-leucine into proteins of rat brain cell structures during application of antifeins (compounds of alternative action on memory processes) has been studied. No correlation was observed between changes in protein synthesis in nuclei, mitochondria, components of endoplasmic reticulum and memory effects of ethyl-, allyl- and propylnorantifeins. Only M1 and M2-demethylated structural analogs of ethylnorantifeins (exerting the most effective action on RNA synthesis and retention of conditional reflexes) enhanced the synaptosomal protein synthesis.

Protein kinase CK2 and regulation of Ca2+-ATPase activity in brain neuron chromatin in rats during aging.

Protein kinase CK2 from neuronal chromatin phosphorylates myosin-like proteins isolated from rat brain chromatin. The protein kinase CK2 activator 1-ethyl-4,5-di(N-methylcarbamoyl)imidazole in vitro stimulated phosphorylation of myosin-like proteins and increased myosin-type Ca(2+)-ATPase activity in neuronal chromatin. After systemic administration this agent normalized Ca(2+)-ATPase activity of brain chromatin in aged rats.

[Effects of antifeins with different memory effects on cAMP phosphodiesterase, lipid peroxidation and RNA synthesis in rat brain neurons]. / Vliianie antifeinov s razlichnymi mnesticheskimi éffektami na fosfodiésterazy tsAMF, perekisnoe okislenie lipidov i sintez RNK v neironakh golovnogo mozga krys.

It has been shown that ethylnorantifein and its structural analogues with opposite effects on long term memory reduce the activity of membrane bound phosphodiesterase cAMP with high and low affinity and exert the same directed influence on lipids peroxidation in membranes. A positive correlation was observed only between the action of these substances on the long term memory and their influence on the RNA synthesis in the rat brain nuclei. Ethylnorantifein and its demethylated analogues increased RNA synthesizing activity while allyl- and propylnorantifeins decreased it. The molecular mechanisms of memory effects of neuroactive substances are discussed.

[State of the "protein kinase CK2-HMG14" system in age-dependent amnesia in rats]. / Sostoianie sistemy "proteinkinaza CK2-HMG14" pri vozrastnoi amnezii u krys.

It has been shown that a decrease in HMGs transcription factors phosphorylation by protein kinase CK2 may be the cause of a gene expression decline in cognitive disorders. Passive avoidance amnesia in old rats (24 month) was accompanied by a decrease in synaptosomal protein synthesis and transcription in isolated nuclei of cortex, hippocampus, and striatum. A decrease in chromatin protein kinase CK2 activity and a significant decrease in HMG14 phosphorylation by CK2 was found in old rats. CK2 selective activators, a 4-carbamoyl-5-N-methylcarbamoyl-1-ethyl-imidazole and 4,5-dicaramoyl-1-ethyl-imidazole, produced the HMG14 phosphorylation and transcription activation in old rats. At the same time, synaptosomal protein synthesis activation and passive avoidance amnesia reduction were observed in old rats. Thus, activation of CK2-HMG14 was accompanied by synaptic plasticity optimisation. The data show a high therapeutic potential of activators of CK2-HMG14.