Influence of calcium on phosphorylation of a synaptosomal protein

Synaptosomal proteins isolated from rat cerebral cortex were phosphorylated endogeneously in the presence of [γ-³²P]ATP. The phosphorylated proteins were found to be membrane bound by differential and density gradient centrifugation. In contrast to the phosphorylation of all synaptosomal proteins, phosphorylation of one protein (C), 41 000–43 000 daltons, was inhibited by Mg²⁺ and stimulated by Ca²⁺. In addition, the ionophores X537A and A23187, as well as papaverine, selectively enhanced phosphorylation of protein C without affecting phosphorylation of the other proteins. Cyclic AMP did not influence the phosphorylation of protein C but markedly affected the phosphorylation of other synaptosomal proteins. It appears that the phosphorylation of protein C is stimulated by agents which trigger the release of neurotransmitters (Ca²⁺, X537A, A23187 and papaverine), and is inhibited by Mg²⁺, which inhibits release. It is proposed that the phosphorylation of protein C is related to membranal events underlying the release of neurotransmitters.     

L-triiodothyronine differentially and nongenomically regulates synaptosomal protein phosphorylation in adult rat brain cerebral cortex: role of calcium and calmodulin

Adult-onset thyroid disorders in humans impair several important central nervous system functions, causing various neuropsychiatric diseases. However, the mechanisms of thyroid hormone (TH) action in the mature mammalian brain remain unclear. Recent nongenomic actions of TH in adult brains are spotlighted. Many nongenomic mechanisms are modulated by phosphorylation-dephosphorylation of substrate proteins. In the present study, L-triiodothyronine (L-T3) demonstrated differential regulation of phosphorylation status of five different synaptosomal proteins (63, 53, 38, 23, and 16 kD) in both a Ca(2+)/calmodulin (CaM)-dependent and -independent manner. L-T3 increased the level of phosphorylation of all these five proteins. Ca(2+)/CaM further stimulated phosphorylation of 63- and 53-kD proteins by L-T3, which were inhibited both by EGTA (Ca(2+)-chelator) or KN62 (Ca(2+)/CaM kinase-II [CaMK-II] inhibitor), suggesting the role of CaMK-II. L-T3 increased the phosphorylation of 23- and 38-kD proteins; the effect was independent of EGTA or KN62. The presence of Ca(2+) decreased L-T3-induced phosphorylation of 63-, 53- and 38-kD proteins. Surprisingly, l-T3-induced phosphorylation of 16-kD protein was not augmented further with Ca(2+) or Ca(2+)/CaM; instead, the presence of CaM abolished the L-T3-induced phosphorylation. EGTA or KN62 could not restore the effect of CaM-induced dephosphorylation of this protein. This study identified the role of Ca(2+)/CaM in the regulation of L-T3-induced protein phosphorylation and supported a unique nongenomic mechanism of second messenger-mediated regulation of protein phosphorylation by TH in mature rat brain. This has profound implications for higher mental functions and strategies for novel therapeutics.     

Non-genomic effect of L-triiodothyronine on calmodulin-dependent synaptosomal protein phosphorylation in adult rat cerebral cortex

The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 microM L-T3, respectively. Chelation of Ca2+ using ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.     

Role of calcium on protein phosphorylation in collagen-stimulated platelets. Effect of a new dihydropyridine derivative

We investigated the effect of extracellular calcium on protein phosphorylation stimulated by collagen in rabbit platelets. We found that collagen-induced increase in 40 kDa protein phosphorylation was maximum at 2 mM Ca2+, and was evident in buffer with zero Ca2+ but not in the presence of EGTA. We also studied the effects of a new dihydropyridine derivative, which has antithrombotic properties, on protein phosphorylation induced by collagen. This compound inhibited the phosphorylation of 40 kDa and 20 kDa protein independently of the extra-cellular Ca2+. The inhibitory effect was dose-dependent but not time-dependent and was more evident when the drug was added before or simultaneously with collagen.     

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation.

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.     

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.     

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.     

Effects of opiates on synaptosomal calmodulin and calcium uptake

Acute opiate administration in vivo increases the level of cytoplasmic calmodulin in isolated rat brain synaptosomes. These synaptosomes do not, however, display decreased K⁺-stimulated⁴⁵Ca uptake in vitro. Opiates affect neither cytoplasmic calmodulin nor Ca uptake after incubation of synaptosomes with the drugs in vitro. In contrast to the interpretation of electrophysiological data, these results suggest that the observed inhibition by opiates of the release of several transmitters may not be mediated by presynaptic opiate receptors that inhibit Ca uptake.     

Role of calcium in synaptosomal substrate oxidation

The effect of veratridine-mediated depolarization on rat brain synaptosomal respiration in the presence and absence of calcium was investigated. Studies on respiration were performed employing three different pretreatments of the synaptosomes which attempted to deplete endogenous substrates. First, synaptosomes were preincubated for 10 min in the absence of any substrates in medium either containing or devoid of calcium. Second, synaptosomes were preincubated for either 15 or 60-min periods in the presence and absence of calcium, and the incubation medium was changed by centrifugation and resuspension of synaptosomes in their respective media. Irrespective of the prior treatment, maximal stimulation of respiration (400-600%) during veratridine (100 microM) elicited depolarization was observed only when calcium was present in the incubation media. In incubations performed in the absence of calcium, veratridine addition either modestly stimulated (10- and 15-min preincubated synaptosomes) or did not affect (60-min preincubated synaptosomes) the rate of respiration. However, when calcium was added back to these incubations the rate of respiration in the presence of veratridine was stimulated by five- to six-fold. Similarly, the rates of 14CO2 production from [1-14C]- and [2-14C]pyruvate were increased by veratridine only when synaptosomes were incubated in calcium-replete medium. These data indicate that calcium plays an obligatory role in depolarization-elicited stimulation of synaptosomal oxidative processes.     

Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22000 dalton protein.

Increases in protein kinase-catalyzed phosphorylation of a 22000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of less than 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.     

Non-requirement of calcium on protamine phosphorylation by calcium-activated, phospholipid-dependent protein kinase

Phosphorylation of clupeine sulfate by purified rat brain calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was studied. In the absence of Ca2+, phosphatidylserine and diolein markedly stimulated its phosphorylation. However Ca2+ did not stimulate but inhibit this phosphorylation about 30% in the presence of phospholipids. Random polymer (Arg, Ser) 3:1 and (Lys, Ser) 3:1 could be phosphorylated by protein kinase C. In the presence of phospholipids Ca2+ is not needed for the phosphorylation of polymer (Arg, Ser) 3:1, while Ca2+ is necessary for polymer (Lys, Ser) 3:1. Non-requirement of Ca2+ on clupeine phosphorylation by protein kinase C is briefly discussed.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Interactions of neomycin and calcium in synaptosomal membranes and polyphosphoinostide monolayers.

Neomycin and related aminoglycosidic antibiotics displace calcium from synaptosomes of guinea pig cerebral cortex and from preparations of phosphatidylinositol diphosphate. At low drug concentrations, inhibition of synaptosomal calcium binding is competitive (Ki = 3-10(-5) M), at high concentrations it is non-competitive (Ki = 4-10(-4) M). Monomolecular films of phosphatidylinositol diphosphate are contracted by low concentrations of neomycin in the subphase, and are expanded at high concentrations. This expansion perists even at the collapse pressure indicating a strong interaction between the drug and the lipid.     

Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low K(m) Ca2+-activated (PDE1C) and Ca2+-independent, rolipram-sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP-dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT-cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT-cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope-tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.     

Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells.

Proliferation of mammalian cells can be controlled by low cultivation temperature. However, depending on cell type and expression system, varying effects of a temperature shift on heterologous protein production have been reported. Here, we characterize growth behavior and productivity of the Chinese hamster ovary (CHO) cell line XM111-10 engineered to synthesize the model-product-secreted alkaline phosphatase (SEAP). Shift of cultivation temperature from 37 degrees C to 30 degrees C caused a growth arrest mainly in the G1 phase of the cell cycle concomitant with an up to 1.7-fold increase of specific productivity. A low temperature cultivation provided 3.4 times higher overall product yield compared to a standard cultivation at 37 degrees C. The cellular and molecular mechanisms underlying the effects of low temperature on growth and productivity of mammalian cells are poorly understood. Separation of total protein extracts by two-dimensional gel electrophoresis showed altered expression levels of CHO-K1 proteins after decrease in cultivation temperature to 30 degrees C. These changes in the proteome suggest that mammalian cells respond actively to low temperature by synthesizing specific cold-inducible proteins. In addition, we provide the first evidence that the cold response of mammalian cells includes changes in postranslational protein modifications. Two CHO proteins were found to be phosphorylated at tyrosine residues following downshift of cultivation temperature to 30 degrees C. Elucidating cellular events during cold exposure is necessary for further optimization of host-cell lines and expression systems and can provide new strategies for metabolic engineering.     

Analysis of protein phosphorylation on a proteome-scale

Phosphorylation, the most intensively studied and common PTM on proteins, is a complex biological phenomenon. Its complexity manifests itself in the large numbers of proteins that attach it, remove it and recognise it as a protein code. Since the first report of protein phosphorylation on vitellin 100 years ago, a wide variety of biochemical and analytical chemical approaches have been developed to enrich and detect protein phosphorylation. The last 5 years have witnessed a renaissance in methodologies capable of characterising protein phosphorylation on a proteome-scale. These technological advances have allowed identification of hundreds to thousands of phosphorylation sites in a proteome and have resulted in a profound paradigm shift. For the first time, using quantitative MS, the topology and significance of global phosphorylation networks may be investigated, marking a new era of cell signalling research. This review addresses recent technological advances in the purification of phosphorylated proteins and peptides and current MS-based strategies used to qualitatively and quantitatively probe these enriched phosphoproteomes. In addition, we review the application of complementary array-based technologies to derive signalling networks from kinase-substrate interactions and discuss future challenges in the field.     

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

吗啡和血管紧张素II对大鼠脑突触小体Ca^2＋摄取的拮抗效应

Behavioral observations have repeatedly shown that the analgesic effect of morphine can be antagonized by intracerebroventricular injection of angiotensin I (A I), although mechanisms underlying the action were obscure. Since a prevention of Ca2+ uptake into the nerve terminals was considered as one of the mechanisms for morphine analgesia, we examined the effect of A I and morphine on the 45Ca uptake by rat brain synaptosomal preparations. Morphine of 10(-8)-10(-6) mol/L produced a dose-related suppression on synaptosomal 45Ca uptake, which was completely reversed by the opioid antagonist naloxone of 10(-6) mol/L. A I of 10(-8)-10(-6) mol/L, on the contrary, enhanced 45Ca uptake. This effect was totally abolished by saralasin, a A I antagonist, at 10(-6) mol/L. When synaptosomal preparations were incubated in a mixture of morphine (10(-6) mol/L) and A I (10(-8)-10(-6) mol/L), the effect of morphine was almost completely reversed. The results suggest that the distinct effect of A I may account for, at least in part, the antagonistic effect of A I on morphine analgesia.     

An endogenous membrane-bound protein inhibits the phosphorylation of the L-type calcium channel by the cAMP-dependent protein kinase.

An endogenous protein inhibits the PKA-phosphorylation of the DHP-binding calcium channel complex in vitro. The inhibitory activity could be reduced by a treatment with detergents or dithiothreitol. Further purification separates the inhibitory activity from the dihydropyridine-binding calcium channel complex. Both activities are localized in the plasma membrane indicating that this protein kinase-inhibitor could interfere with the phosphorylation of the calcium channel by the cAMP-dependent protein kinase. The inhibitory activity may therefore take part in the regulation of the calcium channel current.