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A Routtenberg

Publications and source records attributed to A Routtenberg.

At least 73 records · Page 4Linked to original sources
PubMed1987

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+.

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.

Animals↗
PubMed1987

Gradients of protein kinase C substrate phosphorylation in primate visual system peak in visual memory storage areas.

Two protein kinase C (PKC) substrates of 50 and 81 kDa display topographical gradients in 32P-incorporation along the occipitotemporal visual processing pathway in rhesus monkey cerebral cortex. The 50 kDa protein appears to be homologous to protein F1 from rat (47 kDa) on the basis of isoelectric point, two-dimensional phosphopeptide maps, and kinase specificity, while the 81 kDa protein is probably the same as a previously described PKC substrate. The phosphorylation of protein F1 and 81 kDa was significantly higher in temporal regions of the occipitotemporal pathway, which have been implicated in the storage of visual representations, than in occipital regions, which appear to be less important for visual memory functions. These results suggest that the PKC phosphorylation system, which has been related previously to changes in neural plasticity, plays a progressively greater role in later stages of visual processing, and that this role may involve the storage of visual information in inferotemporal cortical areas.

Animals↗
PubMed1987

Separable roles of hippocampal granule cells in forgetting and pyramidal cells in remembering spatial information.

To investigate the roles individual hippocampal cell groups play in processing of spatial information for memory, we administered low-intensity electrical stimulation to the granule cells, CA3 and CA1 pyramidal cells of the dorsal hippocampus at selected times before and after acquisition of the solution to a radial maze win-stay task. Stimulation of any of the 3 cells populations yielded a variable duration anterograde disruption of memory performance, while stimulation of dentate gyrus granule cells alone produced a declarative memory-specific retrograde amnesia. The amnestic effect of granule cell stimulation was not associated with after discharges in the hippocampus and was prevented by systemic administration of the opiate antagonist naloxone. Our results support the view that this electrical stimulus does not disrupt, but rather, activates the normal function of the granule cell system, resulting in erasure of information held in declarative memory. In contrast, similar activation of the pyramidal cell system does not yield retrograde amnesia, suggesting a normal role for these cells in promoting memory for spatial information.

Animals↗
PubMed1987

Enhancement of long-term potentiation by cis-unsaturated fatty acid: relation to protein kinase C and phospholipase A2.

Previous correlative and interventive work from this laboratory has suggested that activation of protein kinase C (PKC) is important for the maintenance of the hippocampal long-term potentiation (LTP) response. One such study demonstrated that application of the cis-unsaturated fatty acid, oleate, a newly discovered PKC activator, could prolong the time course of LTP. The present study explored the mechanism of cis-unsaturated fatty acid action on LTP produced by perforant path stimulation. First, neither oleate application nor high-frequency stimulation alone produced a persistent change in synaptic transmission, while the 2 in conjunction did so. This suggests that oleate acts synergistically with the consequences of this stimulation to produce an enhancement of the LTP response. Second, oleate enhancement of LTP was more potent when applied in the perforant path synaptic terminal zone than in the dentate hilus, implying that the site of oleate action is at the synapse (where PKC is reported to be enriched). Third, translocation of PKC activity to the membrane was significantly increased after oleate-enhanced LTP relative to vehicle controls. PKC translocation was found to be unaltered by oleate application alone. Fourth, mepacrine blockade of the Ca2+-dependent enzyme phospholipase A2, which releases endogenous oleate from membrane phospholipids, inhibited the time-course of a persistent LTP response. This inhibition was shown to be reversible with oleate application. We propose that high-frequency stimulation produces an elevation of intracellular Ca2+, which then triggers phospholipase A2-mediated oleate release. This free oleate then could act in synergy with processes that render PKC oleate-sensitive to produce a persistent activation of PKC, which is critical for and leads to the persistence of the LTP response.

Animals↗
PubMed1987

Calcium-promoted translocation of protein kinase C to synaptic membranes: relation to the phosphorylation of an endogenous substrate (protein F1) involved in synaptic plasticity.

The translocation of protein kinase C between membrane and cytosol has been implicated in several cellular processes (Kraft and Anderson, 1983; Wooten and Wrenn, 1984; Akers et al., 1985, 1986; Hirota et al., 1985; Wolf et al., 1986). We desired to identify potential trigger mechanisms underlying the translocation of protein kinase C activity to neural membranes following the synaptic plasticity observed after long-term potentiation (LTP; Akers et al., 1986). Takai et al. (1979) have suggested an important role for calcium in protein kinase C translocation; we have therefore studied the effects of Ca2+ on both the translocation of protein kinase C activity and the in vitro phosphorylation of its endogenous substrate, protein F1, in rat hippocampal synaptosomes. Since identical free Ca2+ levels were maintained in subsequent assays of synaptosomal membranes (SPM) and cytosol preparations, alterations in endogenous enzyme activity and in vitro phosphorylation were due to the Ca2+ present during treatment of synaptosomes, and not to the Ca2+ present during assays of enzymatic activity. This afforded the opportunity to relate directly such enzyme translocation to endogenous substrate phosphorylation. The major findings were as follows: 1. Following treatment of synaptosomes with Ca2+, protein kinase C activity in synaptic membrane and protein F1 in vitro phosphorylation were elevated in a dose-dependent manner. 2. The greatest increment in membrane protein kinase C activity and protein F1 in vitro phosphorylation occurred when Ca2+ was increased from 0.1 to 1.0 microM. Maximal levels of enzyme activity were seen following treatment with 10 microM Ca2+, and minimum levels were observed following treatment with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Phospholipid and fatty acid regulation of signal transduction at synapses: potential role for protein kinase C in information storage.

Protein kinase C has been proposed by the author to play a pivotal role in the storage of information. Its activation is directly related to synaptic potentiation as is phosphorylation of certain substrates such as protein F1. Protein F1 is found in highest concentration in those regions of temporal cortex critical for storage of information. The possibility is raised that pathology of this protein kinase C/protein F1 endogenous phosphorylation system may lead to or is responsible for the memory loss of Alzheimer's disease.

GAP-43 Protein↗
PubMed1987

Evidence for the coidentification of GAP-43, a growth-associated protein, and F1, a plasticity-associated protein.

GAP-43 is a fast-axonally transported protein whose expression correlates with periods of axon growth both during development and during regeneration. Similarities in molecular weight (43-47 kDa), pI (4.3-4.5), and aberrant behavior in acrylamide gels suggested that GAP-43 might be related or identical to protein F1, a protein kinase C substrate that has been shown to undergo a change in phosphorylation state during long-term potentiation in the hippocampus. Here we show that GAP-43 and protein F1 comigrate by two-dimensional PAGE and that antiserum raised against GAP-43 specifically immunoprecipitates protein F1. More direct evidence that GAP-43 and protein F1 are identical proteins was obtained by performing S. aureus V8 protease digests of a mixture of purified 32P-labeled protein F1 and purified GAP-43. Under these conditions, 2 phosphorylated peptide fragments of protein F1 corresponded exactly to 2 Coomassie-stainable bands from purified GAP-43. We conclude on the basis of these data that GAP-43 and protein F1 are identical proteins. Using light-microscopic immunocytochemistry, we also show that GAP-43/protein F1 immunoreactivity is localized to neuropil areas of the hippocampus consistent with its roles as a protein kinase C substrate in vivo and in long-term potentiation. These findings suggest that nerve growth during development and regeneration, and synaptic plasticity in the adult mammalian brain, may be mediated by a common mechanism involving the phosphorylation of GAP-43/protein F1.

Animals↗
PubMed1986

Direct relation of long-term synaptic potentiation to phosphorylation of membrane protein F1, a substrate for membrane protein kinase C.

One hour after long-term potentiation (LTP) in the intact hippocampus, a selective increase in protein F1 in vitro phosphorylation was observed in homogenate prepared from dorsal hippocampus. Protein F1 phosphorylation was directly related to the magnitude and persistence of potentiation. No other phosphoprotein studied exhibited a relationship with synaptic enhancement. Low-frequency, non-potentiating stimulation did not increase protein F1 phosphorylation, and phosphorylation of F1 was not elevated when high-frequency stimulation did not produce potentiation. We also confirmed our earlier demonstration of a similar pattern of results 5 min after LTP. In related work we have previously observed: that protein F1 is a substrate for protein kinase C (PKC); that membrane PKC activity was increased by translocation from the cytosol following LTP; and that membrane PKC activity was directly related to the persistence of enhancement. We therefore predicted in the present study that protein F1 phosphorylation in a dorsal hippocampal membrane fraction would be related to LTP. Hippocampal membrane protein F1 was found to be directly related to both the magnitude and persistence of response enhancement. Thus the molecular events leading to prolonged potentiation may involve increased PKC/protein F1 association. Persistence of potentiation may be related to synaptic growth processes involving the growth-associated function of protein F1.

Animals↗
PubMed1986

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids.

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.

Animals↗
PubMed1986

A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus.

Protein kinase C can be activated by oleate, an unsaturated fatty acid. Since protein kinase C is activated by long-term potentiation, we wished to determine whether iontophoretic ejection of oleate into the intact hippocampal dentate gyrus of urethane-anesthetized rats would cause an enhancement of the response potentiated by high frequency stimulation of the perforant path. Oleate ejection did significantly enhance the persistence of the potentiated response. Moreover, a growth of the response beyond the initial potentiation was seen. Arachidonate, which stimulates protein kinase C to a lesser degree, had a significant preservation effect, but no effect on growth of the response. After vehicle and elaidate (trans-stereoisomer of oleate) ejections, the potentiated response decayed to baseline values. In addition, the persistence of the potentiated response observed two hours after its induction was positively correlated with the ability of an unsaturated fatty acid to activate protein kinase C in vitro. The present results support the proposal that protein kinase C activation enhances synaptic strength. It is suggested that one mechanism for this activation may be PLA2-mediated release of oleate.

Animals↗
PubMed1986

Phorbol ester promotes growth of synaptic plasticity.

Iontophoretic application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to the intact rat hippocampus enhances potentiation produced by subsequent high frequency stimulation of the perforant path. The decay of the enhanced population spike amplitude recorded in the hilar dentate gyrus was prevented in animals receiving ejections of TPA, as compared to controls which decayed to baseline values within 2 h following high frequency stimulation. In fact, growth of the potentiated response was observed beginning at 45 min. Similar results were observed with the population excitatory postsynaptic potential slope, a measure of the synaptic response. Since tumour-promoting phorbol esters are known to translocate and activate protein kinase C (PKC) and PKC is translocated to the membrane following hippocampal potentiation, a role for membrane-associated PKC in the regulation of synaptic plasticity is suggested.

Action Potentials↗
PubMed1986

Translocation of protein kinase C activity may mediate hippocampal long-term potentiation.

Protein kinase C activity in rat hippocampal membranes and cytosol was determined 1 minute and 1 hour after induction of the synaptic plasticity of long-term potentiation. At 1 hour after long-term potentiation, but not at 1 minute, protein kinase C activity was increased twofold in membranes and decreased proportionately in cytosol, suggesting translocation of the activity. This time-dependent redistribution of enzyme activity was directly related to the persistence of synaptic plasticity, suggesting a novel mechanism regulating the strength of synaptic transmission.

Animals↗
PubMed1986

Phosphoprotein F1: purification and characterization of a brain kinase C substrate related to plasticity.

To study the role of protein kinase C (PKC) and its substrates in neuronal function, we have investigated the in vitro endogenous phosphorylation of the neuronal phosphoprotein F1 after induction of synaptic plasticity by long-term potentiation (LTP). The protein F1 phosphorylation was found to increase 5 min (Routtenberg et al., 1985), 1 hr (Lovinger et al., 1986) and 3 d (Lovinger et al., 1985) after LTP. The characteristics of this protein bear close similarities to a number of proteins characterized in various neuronal systems, such as B50 (brain specific, synaptosome-enriched protein), pp46 (a growth cone protein), and GAP 43 (nerve growth and regeneration-associated protein). A positive identification of the purified protein F1 with these proteins would link protein F1 to the developmental growth of axons, nerve regeneration, and polyphosphoinositide metabolism, as well as adult plasticity. We have therefore purified and partially characterized native protein F1 so that a meaningful comparison among the properties of these proteins can be made. Using synaptosomal plasma membrane (P2') as starting material, subsequent purification involved pH extraction, 40-80% ammonium sulfate precipitation, hydroxylapatite, and phenyl-Sepharose column chromatography. This procedure achieved greater than 800-fold purification and about 45% yield relative to P2'. Purified protein F1 (Mr = 47,000, pI = 4.5) was found to be a hydrophilic molecule and was phosphorylated by 1000-fold purified PKC in the presence of phosphatidylserine (PS) and Ca2+. The Ka of PS activation is about 15 micrograms/ml (approximately 20 microM), and that of Ca2+ is about 25 microM. Diolein and DiC:8 (a synthetic diacylglycerol) lowered the requirement of Ca2+ for maximal stimulation from 100 to 5 microM. Ca2+-calmodulin kinases type I and II did not phosphorylate protein F1. The phosphoamino acid analysis showed that 97% of the total incorporated 32P-phosphate was on the serine residue. Phosphopeptide mapping using V8-protease generated 2 phospho-fragments having apparent Mr of 13,000 and 11,000. Calmodulin at 3.6 microM inhibited 95% of protein F1 phosphorylation by PKC. The availability of purified native protein F1 should facilitate investigation of the physiological role of this protein in the nervous system and its functional regulation by PKC.

Animals↗
PubMed1985

Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2+.

Unsaturated fatty acids (oleic acid and arachidonic acid) activate purified protein kinase C independently of phospholipid and Ca2+. Oleic acid activation of protein kinase C is as effective as phosphatidylserine and Ca2+. Ka values for oleic acid and arachidonic acid are 50 and 53 microM, respectively. In contrast to the cis fatty acids, a trans form (elaidic acid) or a saturated fatty acid (stearic acid) has little or no effect on protein kinase C activation. If cis fatty acid liberation is physiologically important, this suggests that another mechanism may exist for protein kinase C activation, in addition to phospholipase C/phosphatidylinositol turnover signaling, possibly via the liberation of cis fatty acids by the Ca2+-dependent phospholipase A2 system.

Animals↗
PubMed1985

A selective increase in phosporylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement.

Increased in vitro phosphorylation of the 47 kdalton, 4.5 pI protein F1 was observed in dorsal hippocampal tissue from animals exhibiting long term enhancement (LTE) three days after high frequency stimulation of the perforant pathway, as compared to tissue from low frequency stimulated controls or from unoperated animals. The increase in protein F1 phosphorylation was related to LTE rather than simple activation of perforant path-dentate gyrus synapses. This is the first report of a change in brain protein phosphorylation accompanying synaptic enhancement lasting days. The extent of growth of LTE over the three days following stimulation was directly related (r = +0.66, P less than 0.05) to protein F1 phosphorylation. Among the phosphoproteins studied this relationship between LTE and phosphorylation was selective for protein F1. This suggests that protein F1 may regulate growth of synaptic plasticity for at least a three day period. The mechanism for the LTE-related increase in protein F1 phosphorylation has not been established. However, recent evidence from this laboratory indicates: that protein F1 is phosphorylated by the calcium/phospholipid-dependent protein kinase C; and that kinase C is activated 1 h after LTE. Therefore, the increase in protein F1 phosphorylation following LTE may result from long term activation of protein C kinase.

Animals↗