Repeated amphetamine treatment increases phosphorylation of extracellular signal-regulated kinase, protein kinase B, and cyclase response element-binding protein in the rat striatum

Extracellular signal-regulated kinases, protein kinase B/Akt and cyclase response element-binding protein play important roles in drug-induced neuroadaptations. Acute psychostimulant exposure rapidly alters the phosphorylation of these proteins in the striatum but less is known about their responses to repeated stimulant administration. In this study the phosphorylated state of these proteins in rat striatum was analyzed by immunoblotting 15 min and 2 h after amphetamine (AMPH)-induced behavioral sensitization. Two weeks after the last dose of 5 mg/kg, i.p. AMPH once daily for 5 days, rats were challenged with 1 mg/kg, i.p. AMPH or saline and sacrificed 15 min or 2 h later. Sensitization to AMPH-induced behavioral activity was observed in AMPH pre-treated rats after AMPH on the challenge day. Phosphorylation of all three proteins was significantly greater 15 min after AMPH in AMPH-pre-treated than in saline-pre-treated rats. Two hours after AMPH challenge in AMPH-pre-treated rats, phospho-extracellular signal-regulated kinase and phospho-cAMP response element-binding protein immunoreactivity was still significantly elevated but not after AMPH injection in saline-pre-treated rats. In contrast, phospho-Akt was down-regulated to the same extent 2 h after acute AMPH or repeated AMPH with an AMPH challenge. These data implicate differential regulation of phospho-extracellular signal-regulated kinase, phospho-cAMP response element-binding protein versus phospho-Akt in sensitized responses to AMPH.     

cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Evidence of Abeta- and transgene-dependent defects in ERK-CREB signaling in Alzheimer's models

Extracellular-signal regulated kinase (ERK) signaling is critical for memory and tightly regulated by acute environmental stimuli. In Alzheimer disease transgenic models, active ERK is shown to first be increased, then later reduced, but whether these baseline changes reflect disruptions in ERK signaling is less clear. We investigated the influence of the familial Alzheimer's disease transgene APP sw and β-amyloid peptide (Aβ) immunoneutralization on cannulation injury-associated (i.c.v. infusion) ERK activation. At both 12 and 22 months of age, the trauma-associated activation of ERK observed in Tg⁻ mice was dramatically attenuated in Tg⁺. In cortices of 22-month-old non-infused mice, a reduction in ERK activation was observed in Tg⁺, relative to Tg⁻ mice. Intracerebroventricular (i.c.v.) anti-Aβ infusion significantly increased phosphorylated ERK, its substrate cAMP-response element-binding protein (CREB) and a downstream target, the NMDA receptor subunit. We also demonstrated that Aβ oligomer decreased active ERK and subsequently active CREB in human neuroblastoma cells, which could be prevented by oligomer immunoneutralization. Aβ oligomers also inhibited active ERK and CREB in primary neurons, in addition to reducing the downstream post-synaptic protein NMDA receptor subunit. These effects were reversed by anti-oligomer. Our data strongly support the existence of an APP sw transgene-dependent and Aβ oligomer-mediated defect in regulation of ERK activation.     

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.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 - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - PDPK proline-dependent protein kinase     

A low molecular weight substance purified from human placenta inhibits cAMP-dependent protein kinase and activates protein kinase C

We have purified from human placenta a low molecular mass substance that inhibits cAMP-dependent protein kinase and activates protein kinase C. This protein kinase regulator was purified in three steps: (1) homogenizing placentas in chloroform/methanol and extracting the regulator into water; (2) eluting a strong anion exchange high performance liquid chromatography (HPLC) column with a quaternary gradient; and (3) eluting a reversed-phase HPLC column with a binary gradient. The regulator was found to be highly purified by HPLC, thin-layer chromatography (TLC) and laser desorption ionization mass spectrometry with a molecular mass of 703 Daltons by the latter procedure. The physical and biochemical properties of this protein kinase regulator suggest that it is a phospholipid but it did not co-elute by HPLC or by TLC with any of the known phospholipid activators of protein kinase C.     

Induction of apoptosis by a calpain stimulator, ONO-3403

The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 g/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 g/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified -calpain at a concentration higher than 100 g/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of -calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro

Dipyridamole activates in vitro type II CAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of CAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP 2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A₁ receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883–888, 1989) might be mediated through a direct action — an activation — on the catalytic subunit of a cAMP-dependent protein kinase.     

Ubiquilin-1 and protein quality control in Alzheimer disease

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Altered cAMP-dependent protein kinase subunit immunolabeling in post-mortem brain from patients with bipolar affective disorder

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP-dependent protein kinase (PKA) in discrete post-mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform-specific polyclonal antisera, levels of PKA RIalpha, RIbeta, RIIalpha, RIIbeta and Calpha subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post-mortem brain from BD patients and matched, non-neurological, non-psychiatric controls. Immunoreactive levels of cytosolic Calpha in temporal and frontal cortices, as well as that of cytosolic RIIbeta in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post-mortem interval or pH and unrelated to ante-mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up-regulated in discrete cerebral cortical regions in BD.     

Inhibition of 2'-phosphodiesterase by cAMP-dependent protein kinase. Involvement of phosphorylation of protein inhibitor

2'-Phosphodiesterase from NIH 3T3 cells was purified about 530-fold. Treatment of the cell lysate with the cAMP-dependent protein kinase causing the 2'-phosphodiesterase inhibition did not result in phosphorylation of the enzyme itself. The kinase was found to phosphorylate a specific 18-kDa protein, the phosphorylated form of this protein being the inhibitor of 2'-phosphodiesterase.     

The unsolved relationship of brain aging and late-onset Alzheimer disease

Late-onset Alzheimer disease is the most common form of dementia and is strongly associated with age. Today, around 24 million people suffer from dementia and with aging of industrial populations this number will significantly increase throughout the next decades. An effective therapy that successfully decelerates or prevents the progressive neurodegeneration does not exist. Histopathologically Alzheimer disease is characterized by extensive extracellular amyloid β (Aβ) plaques, intracellular neurofibrillary tangles (NFTs), synaptic loss and neuronal cell death in distinct brain regions. The molecular correlation of Aβ or NFTs and development of late-onset Alzheimer disease needs further clarification. This review focuses on structural and functional alterations of the brain during aging, age-associated imbalances of defences against oxidative stress and age-related alterations of the metabolism of Aβ, via a comparison of observations in healthy aged individuals and cognitively impaired or AD patients. Although our understanding of brain region-specific neuronal aging is still incomplete, the early structural and molecular changes in the transition from cognitive health to impairment are subtle and the actual factors triggering the severe brain atrophy during LOAD remain ambiguous.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Plasma prion protein concentration and progression of Alzheimer disease

Background/Objective: Recently, PrP^c has been linked to AD pathogenesis. Second, a relation of PrP^c plasma levels with cognitive status and decline of healthy elderly subjects has been reported. Therefore, we hypothesized baseline plasma levels of PrP^c to be associated with AD progression in cognitive and functional domains.Materials and Methods: AD patients (n = 84) were included into an observational study at time of diagnosis. Baseline plasma PrP^c levels were determined. Decline was assessed annually (mean follow-up time 3 years) with the aid of different standardized tests (MMSE, iADL, bADL, GDS, UPDRSIII). Multiple regression analyses were used to uncover potential associations between decline and PrP^c levels.Results: No association of PrP^c and decline could be established. Presence of diabetes mellitus was linked to slower deterioration. Intake of neuroleptic drugs or memantine was associated with faster progression.Conclusion: Plasma PrP^c at baseline could not be shown to be related to AD progression in this study. An interesting association of diabetes mellitus and decline warrants further investigation.     

Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.     

Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase

Summary ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive²²Na⁺ uptake and barium-sensitive, voltage-dependent⁸⁶Rb⁺-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive⁸⁶Rb⁺ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P<0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 g/ml) was also present in the vesicle solutions. The stimulation of⁸⁶Rb⁺ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Influence of protein nutrition on calpain activity of mouse kidney: Modulation by calpastatin

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system. (Mol Cell Biochem 166: 95-99, 1997)