Protein Phosphorylation In Brain Research Articles

Dysregulated Brain Protein Phosphorylation Linked to Increased Human Tau Expression in the hTau Transgenic Mouse Model.

Altered protein phosphorylation is a major pathologic modification in tauopathies and Alzheimer’s disease (AD) linked to abnormal tau fibrillar deposits in neurofibrillary tangles (NFTs) and pre-tangles and β-amyloid deposits in AD. hTau transgenic mice, which express 3R and less 4R human tau with no mutations in a murine knock-out background, show increased tau deposition in neurons but not NFTs and pre-tangles at the age of nine months. Label-free (phospho)proteomics and SWATH-MS identified 2065 proteins in hTau and wild-type (WT) mice. Only six proteins showed increased levels in hTau; no proteins were down-regulated. Increased tau phosphorylation in hTau was detected at Ser199, Ser202, Ser214, Ser396, Ser400, Thr403, Ser404, Ser413, Ser416, Ser422, Ser491, and Ser494, in addition to Thr181, Thr231, Ser396/Ser404, but not at Ser202/Thr205. In addition, 4578 phosphopeptides (corresponding to 1622 phosphoproteins) were identified in hTau and WT mice; 64 proteins were differentially phosphorylated in hTau. Sixty proteins were grouped into components of membranes, membrane signaling, synapses, vesicles, cytoskeleton, DNA/RNA/protein metabolism, ubiquitin/proteasome system, cholesterol and lipid metabolism, and cell signaling. These results showed that over-expression of human tau without pre-tangle and NFT formation preferentially triggers an imbalance in the phosphorylation profile of specific proteins involved in the cytoskeletal–membrane-signaling axis.

Dysregulated brain protein phosphorylation linked to increased human tau expression: the hTau transgenic mouse model

Dysregulated brain protein phosphorylation linked to increased human tau expression: the hTau transgenic mouse model

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3β(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3β, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3β (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.

Zebrafish brain proteomics reveals central proteins involved in neurodegeneration

Understanding the complex biology of the brain requires analyzing its structural and functional complexity at the protein level. The large-scale analysis of the brain proteome, coupled with characterization of central brain proteins, provides insight into fundamental brain processes and processes linked to neurodegenerative diseases. Here we provide a map of the zebrafish brain proteome by using two-dimensional gel electrophoresis (2DE), followed by the identification of 95 brain proteins using mass spectrometry (LC-ESI MS/MS). Our data show extensive phosphorylation of brain proteins but less prominent glycosylation. Furthermore, ~51% of the identified proteins are predicted to have one or more ubiquitination sites whereas ~90% are predicted to have one or more SUMOylation sites. Our findings provide a valuable proteome map of the zebrafish brain and associated posttranslational modifications demonstrating that zebrafish proteomic approaches can aid in our understanding of proteins central to important neuronal processes and those associated with neurodegenerative disorders.

Impairment of conditioned freezing to tone, but not to context, in Fyn‐transgenic mice: relationship to NMDA receptor subunit 2B function

We previously demonstrated that transgenic mice overexpressing Fyn tyrosine kinase exhibit higher seizure susceptibility and enhanced tyrosine phosphorylation of several proteins, including the N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B). In the present study, we analysed behavioural phenotypes, especially conditioned fear responses, of Fyn-transgenic (TG) mice to better understand the role of Fyn in learned emotional behaviour. Tone-dependent conditioned freezing was significantly attenuated in Fyn-TG mice, whereas context-dependent freezing was unaffected. Neither massed nor spaced conditioning ameliorated the attenuation of tone-dependent freezing. However, the selective NR2B antagonist ifenprodil, when administered before conditioning, restored tone-dependent freezing in Fyn-TG mice at a dose that did not affect freezing in wild-type (WT) mice. These results suggest that impairment of tone-dependent conditioned freezing in Fyn-TG mice is caused by disruption of the NR2B-containing NMDA receptor function. Tyrosine phosphorylation of brain proteins, including NR2B, was enhanced in Fyn-TG mice compared with that in WT mice. We also found that ifenprodil significantly suppressed the enhanced tyrosine phosphorylation. Thus, our data support the notion that NMDA receptor activity is tightly correlated with protein tyrosine phosphorylation, and Fyn might be one key molecule that controls tone-dependent conditioned freezing through the regulation of NMDA receptor function.

Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: comparison with other methods of sacrifice and analysis of multiple phosphoproteins

At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.

Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain

The reversible protein phosphorylation is the most important cellular regulation of the biological functions of many proteins. Disregulation of protein phosphorylation is involved in pathogeneses of several human diseases. The abnormal hyperphosphorylation of microtubule-associated protein tau and its aggregation into neurofibrillary tangles in selective neurons is one of the major brain pathologies of Alzheimer's disease and several other related neurodegenerative diseases. Here we present metabolically competent rat brain slices as a model to study the regulation of protein phosphorylation in brain. Employing this model we have been able to study the abnormal hyperphosphorylation of tau and other microtubule-associated proteins. We have evaluated the activity and intactness of the rat brain slices both biochemically and morphologically. Selective inhibition of protein phosphatase 2A in these rat brain slices by the treatment with okadaic acid induced hyperphosphorylation of tau at many abnormal sites seen in Alzheimer's disease brain and the accumulation of hyperphosphorylated tau in pyramidal neurons of the cortex and hippocampus. The regulation of the phosphorylation of high-molecular-weight microtubule-associated protein, MAP1b, was also studied with this model. This model enables studies on the regulation of protein phosphorylation not only biochemically, but also histochemically and immunocytochemically. Furthermore, unlike cultured cells, the neurons in the brain slices reside in the physiological environment of the brain consisting of natural extracellular matrix, neuronal connectivity, and neuronal-glial interactions.

Effect of Verapamil on Changes in Brain Protein Phosphorylation after Oral Administration of Tri-O-cresyl Phosphate to Hens

Abstract The endogenous phosphorylation of specific brain proteins in white Leghorn laying hens treated with tri- o -cresyl phosphate (TOCP) with and without verapamil, a calcium channel blocker, which has been shown to ameliorate clinical signs and nervous lesions associated with organophosphate-induced delayed neuropathy (OPIDN), was determined. Verapamil was given im at the dose of 7 mg/kg/day for 4 days beginning 1 day before administration of TOCP (750 mg/kg, po). Phosphorylation of crude synaptosomal (P2) membrane proteins was assayed in vitro using [γ- 32 P]ATP as a phosphate donor. Following resolution of brain membrane proteins by SDS-PAGE, specific protein phosphorylation was detected by autoradiography and quantified by densitometry TOCP caused, 21 days after dosing, an enhancement of the phosphorylation of membrane proteins with apparent molecular weights of 40, 55, and 60 kDa by as much as 253, 322, and 609%, respectively. Verapamil administered to TOCP-treated hens abolished the enhancement and even caused some inhibition. This indicates that calcium channel blocker may contribute to the amelioration of OPIDN by preventing the enhancement of phosphorylation of brain membrane proteins.

Calretinin, a neuronal calcium binding protein, inhibits phosphorylation of a 39 kDa synaptic membrane protein from rat brain cerebral cortex

The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin. At the same concentration, calretinin produced a slight increase in the phosphorylation of several other synaptic membrane proteins which appeared additive with the stimulation produced by either calmodulin or phosphatidylserine in the presence of calcium.

Posthoc phosphorylation of proteins derived from ischemic rat hippocampus, striatum and neocortex

Disruption of the brain's protein phosphorylation system by ischemia may cause irreversible metabolic and structural alterations leading eventually to cell death. To examine the effect of ischemia on the phosphorylation state of brain proteins, tissue homogenates derived from the hippocampus, striatum and neocortex of normal rats and rats subjected to severe forebrain ischemia were phosphorylated with [gamma-32P]ATP. The phosphorylated proteins were separated by two-dimensional polyacrylamide gel electrophoresis and changes were assessed by autoradiography. Cerebral ischemia caused marked alterations of the phosphorylation state of many brain proteins; phosphorylation of some proteins was increased while phosphorylation of others was decreased. Despite differences in the sensitivity of the hippocampus, striatum and neocortex to ischemic injury the direction and approximate magnitude of protein phosphorylation changes caused by ischemia were similar in all three regions. Since the pattern of protein phosphorylation in the ischemia-vulnerable hippocampus was identical to that in the ischemia-resistant paramedian neocortex we conclude that abnormalities of protein phosphorylation may be necessary for ischemic injury to neurons but none are sufficient to explain the selective vulnerability of certain brain regions to ischemic damage.

Pp60 c-src Kinase expression in brain of adult rats in relation to age

Although there is evidence of alterations in brain protein phosphorylation patterns with age, it is not known if the protein kinases that phosphorylate only at tyrosine residues are involved in these changes. For this reason, we examined the age-related expression of pp60c-src, a tyrosine protein kinase enriched in neural tissues, in whole brain of adult Fischer-344 rats. The pp60c-src kinase activity was immunoprecipitated using a monoclonal antibody and the incorporation of [32P] from radiolabeled ATP into an exogenous substrate (casein) measured. The results showed that there was a substantial amount of pp60c-src kinase activity in brain of the adult animals ranging in age from 4 to 23 months and that it was not significantly different among these groups. Also, immunoprecipitates obtained under conditions of monoclonal antibody excess and utilized for immunoblot analysis indicated that the relative levels of the pp60c-src protein were unchanged in the same animals. These results suggest that, at the whole brain level, the pp60c-src kinase has a stable turnover and that a high amount of activity is biologically important in brain of adult rats through early senescence.

Protein phosphorylation systems in postmortem human brain.

Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.

Ganglioside-modulated protein phosphorylation. Partial purification and characterization of a ganglioside-stimulated protein kinase in brain.

Gangliosides have profound modulatory effects on protein phosphorylation in brain. A protein kinase activated directly by gangliosides has been partially purified from the particulate fractions of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, and gel filtration. This novel ganglioside-stimulated protein kinase is distinct from cAMP-dependent, Ca2+/calmodulin-dependent, and Ca2+/phospholipid-dependent protein kinases. The partially purified kinase preparation could undergo ganglioside-stimulated autophosphorylation of a major phosphoprotein with Mr corresponding to 68,000. It also could phosphorylate exogenous substrates such as the synthetic peptide Leu-Arg-Arg-Ala Ser-Leu-Gly. The requirement of gangliosides for the activation of kinase activity is dose-dependent and specific. Among the various gangliosides tested, GT1b and GD1a were found to be the most potent activators, whereas GD1b and GM1 were slightly less effective. The activation process is rapid and does not require the presence of Ca2+, suggesting that the stimulatory effect of gangliosides is not mediated through limited proteolysis or Ca2+-glycolipid complexes. Although the exact physiological significance of the ganglioside-stimulated protein kinase is not known at present, it is possible that certain functions related to gangliosides in the nervous system are mediated through the activation of this novel enzyme.

Decrease in protein phosphorylation in central and peripheral nervous tissues of methylmercury-treated rat.

The protein phosphorylation in extracts of nervous tissues of rats acutely exposed to methylmercury chloride (seven daily injections of 10 mg methylmercury chloride/kg body weight) was examined. In the brain, the phosphorylating activity was dependent on cAMP and Mg2+. The effect of methylmercury on the phosphorylation of brain proteins, including tubulin and MAP-2, was hardly discernible. In peripheral nervous tissues such as the dorsal and ventral roots, sciatic nerves and dorsal root ganglia, the phosphorylating activity was dependent on Ca2+, and the maximal activity was obtained when the tissues were extracted in the presence of 1% Triton X-100. SDS-Polyacrylamide gel electrophoresis revealed that the major phosphorylated proteins in the peripheral tissues were myelin proteins. The effects of methylmercury were not uniform regarding protein species and tissues. The most marked changes were observed in sciatic nerves, in which phosphorylation of the 33 kDa, 28 kDa, 19 kDa, 18 kDa and 15 kDa proteins was significantly decreased in the symptomatic phase of intoxication.

A selective increase in phosphorylation 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.

Effect of vanadate on brain protein phosphorylation.

The effect of vanadate on the phosphorylation of synaptosomal membrane proteins prepared from rat cerebral cortex was studied. Vanadate concentrations of 10(-6), 10(-5), and 10(-4) M increased the endogenous phosphorylation activity by 25%, 37%, and 75%, respectively. Increasing the ATP concentration in the assay medium from 50 to 500 microM did not influence the above effect. A commercial preparation of the purified protein kinase was stimulated 40% by 10(-3) M vanadate. Calcium-calmodulin dependent activity was stimulated only 20% by 10(-5) M vanadate. The effect was not enhanced by further increasing vanadate concentration. Addition of calcium ions (above 50 microM) suppressed the vanadate effect, while an inhibition was observed at high Ca2+ concentration (2.5 mM). Below 50 microM calcium ions stimulated phosphorylation activity in the absence of vanadate and did not affect the stimulatory action of vanadate. Cyclic AMP-dependent endogenous phosphorylation was also stimulated by vanadate. Activation by cAMP could not be observed at vanadate concentrations above 10(-6) M. Possible mechanisms of the vanadate effect are discussed.

Brain protein phosphorylation [formula omitted]: Selective substrate action of insulin

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50 microM Mg++ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein F1. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1 mM Mg++, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein F1 is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca++/phospholipid protein kinase.

S-100-mediated inhibition of brain protein phosphorylation.

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.