Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, alpha-CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP-Dependent Protein Kinase in Rat Frontal Cortex

The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by approximately 25%, whereas enzyme activity was increased in the particulate fractions by approximately 20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant drugs, because representatives of several other classes of psychotropic drugs-namely, haloperidol, morphine, and diazepam--failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the clinical actions of these treatments.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol³²P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,³²P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phosphorylation: a molecular switch in opioid tolerance

Protein phosphorylation is a key posttranslational modification mechanism controlling the conformation and activity of many proteins. Increasing evidence has implicated an essential role of phosphorylation by several major protein kinases in promoting and maintaining opioid tolerance. We review some of the most recent studies on protein kinase C (PKC), cyclic AMP dependent protein kinase A (PKA), calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase G (PKG), and G protein receptor kinase (GRK). These kinases act as the molecular switches to modulate opioid tolerance. Pharmacological interventions at one or more of the protein kinases and phosphatases may provide valuable strategies to improve opioid analgesia by attenuating tolerance to these drugs.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.     

Regulation of Ca2+ influx in myocardial cells by beta adrenergic receptors,cyclic nucleotides,and phosphorylation

Calcium channels in the heart play a major role in cardiac function. These channels are modulated in a variety of ways, including protein phosphorylation. Cyclic AMP-mediated phosphorylation is the best understood phosphorylation mechanism which regulates calcium influx into cardiac cells. Binding of an agonist (e.g., a catecholamine) to the appropriate receptor stimulates production of cyclic AMP by adenylate cyclase. The cyclic AMP may subsequently bind to and activate a cyclic AMP-dependent protein kinase, which then can phosphorylate a number of substrates, including the calcium channel (or a closely-associated regulatory protein). This results in stimulation of the calcium channels, greater calcium influx, and increased contractility. The cyclic AMP system is not the only protein kinase system in the heart. Thus, the possibility exists that other protein kinases may also regulate the calcium channels and, hence, cardiac function. Recent evidence suggests that cyclic GMP-mediated phosphorylation may play a role opposite to cyclic AMP-mediated phosphorylation, i.e., inhibition of the calcium current rather than stimulation. Other recent evidence also suggests that a calcium/calmodulin-dependent protein kinase and calcium/phospholipid-dependent protein kinase (protein kinase C) may also regulate the myocardial calcium channels. Thus, protein phosphorylation may be a general mechanism whereby calcium channels and cardiac function are modulated under a variety of conditions.     

Phosphorylation of τ Protein by Casein Kinase-1 Converts It to an Abnormal Alzheimer-Like State

Abstract: The microtubule-associated protein τ is abnormally hyperphosphorylated in Alzheimer's disease. Both proline-dependent protein kinases (PDPKs) and non-PDPKs are involved in this hyperphosphorylation of τ. Several PDPKs can phosphorylate τ in vitro and induce Alzheimer-like epitopes to many phosphorylation-dependent antibodies. A similar induction has not been reported with non-PDPKs. In this study we have evaluated six non-PDPKs [cyclic AMP-dependent (A-kinase), calcium/phospholipid-dependent (C-kinase), casein kinase-1 (CK-1), casein kinase-2 (CK-2), calcium/calmodulin-dependent protein kinase II, and calcium/calmodulin-dependent protein kinase from rat cerebellum] for their abilities to induce Alzheimer-like epitopes on τ. Such epitopes were induced by A-kinase, C-kinase, CK-1, and CK-2, but the degree of induction achieved by CK-1 was much greater than with the other kinases. These results suggest that CK-1 may play an important role in the conversion of τ from the normal to the abnormal phosphorylation state in Alzheimer's disease.     

Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice

In plants, calcium acts as a universal second messenger in various signal transduction pathways. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles regulating downstream components of calcium signaling. We conducted a genome-wide analysis of rice CDPKs and identified 29 CDPK genes and eight closely related kinase genes, including five CDPK-related kinases (CRKs), one calcium and calmodulin-dependent protein kinase (CCaMK) and two phosphoenolpyruvate (PEP) carboxylase kinase-related kinases (PEPRKs). The mRNA splicing sites of the rice CDPKs, CRKs and PEPRKs (but not OsCCaMK) are highly conserved, suggesting that these kinases are derived from a common ancestor. RNA gel blot analyses revealed that the majority of rice CDPK genes exhibited tissue-specific expression. Expression of OsCPK9 was elevated in seedlings infected by rice blast, indicating that this gene plays an important role in signaling in response to rice blast treatment. Our genomic and bioinformatic analyses will provide an important foundation for further functional dissection of the rice CDPK gene family.     

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.     

Mechanisms of action of antidepressants: from neurotransmitter systems to signaling pathways

Antidepressants are commonly used in the treatment of anxiety and depression, medical conditions that affect approximately 17-20% of the population. The clinical effects of antidepressants take several weeks to manifest, suggesting that these drugs induce adaptive changes in brain structures affected by anxiety and depression. In order to develop shorter-acting and more effective drugs for the treatment of anxiety and depression, it is important to understand how antidepressants bring about their beneficial effects. Recent reports suggest that antidepressants can induce neurogenesis in the adult brain, although the mechanisms involved are not clearly understood. In this review, we describe the different neurotransmitter systems that are affected by anxiety and depression and how they are modulated by antidepressant treatment with a focus on signaling molecules and pathways that are activated during neurotransmitter receptor induced neurogenesis.     

Filopodial behavior is dependent on the phosphorylation state of neuronal growth cones

Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.     

Mediators of the mitogenic action of human V(1) vascular vasopressin receptors

Arginine vasopressin (AVP) activation of V(1) vascular receptors (V(1)Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of V(1)R, Chinese hamster ovary (CHO) cells were stably transfected with the human V(1)R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of V(1)Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and G(2)-M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of V(1)R activation included calcium mobilization, coupling to a G(q) protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulin-dependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

Rapid Activation of the Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Signaling Pathway by Electroconvulsive Shock in the Rat Prefrontal Cortex Is Not Associated with TrkB Neurotrophin Receptor Activation

1. Emerging evidence indicates that brain-derived neurotrophic factor (BDNF) and its receptor TrkB play important roles in the mechanism of action of electroconvulsive shock (ECS) treatment. ECS produces a significant increase in brain BDNF synthesis together with a variety of neuroplastic changes including neurogenesis and axonal sprouting in the rodent brain, which is believed to be associated to the antidepressant effect of ECS. ERK1/2 (extracellular signal-regulated kinase-1/2) and Akt (protein kinase B), both intracellular signaling molecules being linked to neurotrophin signaling and synthesis, are important pathways triggered by TrkB autophosphorylation. 2. We have previously observed that chemical antidepressants induce a rapid activation of TrkB signaling in the rodent prefrontal cortex (PFC), which is likely a consequence of the stimulatory effect of antidepressants on BDNF synthesis. However, it is not known whether ECS triggers TrkB autophosphorylation and if any ECS-induced effect on TrkB function may be associated with the activation of the ERK1/2 and Akt pathways. 3. The present study assayed the phosphorylation levels of TrkB, ERK1/2, and Akt in the PFC of sham and ECS-treated rats. While the TrkB autophosphorylation (pTrkB) levels were decreased 30 min after both acute and chronic ECS, no change in pTrkB levels were observed at any other time points measured. In contrast, acute but not chronic ECS, transiently induced a very rapid and robust hyperphosphorylation of ERK1/2. Akt phosphorylation levels remained unchanged following acute or chronic ECS. Hence, although ECS effectively stimulates the ERK1/2 pathway in the PFC, this effect does not appear to involve upstream activation of TrkB.     

Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.     

Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP

The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.