Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase.

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation.

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

Anhydride modified cantharidin analogues. Is ring opening important in the inhibition of protein phosphatase 2A?

A series of anhydride modified cantharidin analogues have been synthesised and screened for their ability to inhibit protein phosphatase 2A. Surprisingly only analogues capable of undergoing a facile ring opening of the anhydride moiety displayed any significant inhibition. Subsequent NMR experiments indicated that 7-oxobicyclo[2.2.1]heptane-2,3-dicarboxylic acid was the major (sole) species under assay conditions. The ability of these modified anhydro-cantharidin analogues to inhibit protein phosphatase 2A varies from 4 (16) to 100% (8) at 100 microM test concentration.

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc.

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.

Transient translocation and activation of protein phosphatase 2A during mast cell secretion.

Okadaic acid inhibits secretion from mast cells, suggesting a regulatory role for protein Ser/Thr phosphatases type I (PP1) and/or 2A (PP2A) in the secretory process. In unstimulated RBL-2H3 cells, okadaic acid pretreatment inhibited PP2A activity in both cytosol and membrane fractions, but inhibition of secretion correlated with inhibition of membrane-bound rather than cytosolic PP2A activity. Okadaic acid had very little effect on PP1 activity. Stimulation of RBL-2H3 cells by antigen led to the activity and amount of PP2A in the membrane fraction increasing nearly 2-fold. In contrast, there was little change in the activity or distribution of PP1. Importantly, the translocation of PP2A was transient, coinciding with or marginally preceding the peak rate of secretion, suggesting a link between PP2A translocation, activity, and secretion. Phorbol 12-myristate 13-acetate plus the calcium ionophore A23187 induced a slower, prolonged rate of secretion that coincided with a similarly protracted translocation of PP2A to the membrane fraction. PP2A translocation is not the only event required for secretion as translocation was also induced by phorbol 12-myristate 13-acetate, without resulting in secretion. These results indicate that increased protein dephosphorylation in the membrane fraction mediated by PP2A is required for mast cell secretion. To our knowledge, this is the first demonstration of a signal-mediated, rapid, transient translocation and activation of PP2A in membranes in any system.

Targeting of PKA, PKC and protein phosphatases to cellular microdomains.

The intracellular responses to many distinct extracellular signals involve the direction of broad-based protein kinases and protein phosphatases to catalyse quite specific protein phosphorylation/dephosphorylation events. It is now clear that such specificity is often achieved through subcellular targeting of distinct pools of kinase or phosphatase towards particular substrates at specific subcellular locations. Given the dynamic nature of protein phosphorylation reactions, coordinated control of both kinase and phosphatases is often required and complexes formed by common scaffold or targeting proteins exist to direct both kinase and phosphatase to the same subcellular location. In many cases more than one kinase or phosphatase is required and binding proteins which target more than one kinase or phosphatase have now been identified. This review summarizes recent findings relating to the concept of targeting PKA, PKC and the major serine/threonine phosphatases, PP1, PP2A and PP2B, through the formation of multi-enzyme signalling complexes.

Developmental regulation of protein phosphatase types 1 and 2A in post-hatch chicken brain.

The activity and subcellular distribution of protein phosphatases 1 and 2A were measured in chicken forebrain and cerebellum during post-hatch development. At all post-hatch ages, a large proportion of PP1 and PP2A was membrane bound and these enzymes were less active than their cytosolic counterparts. The protein concentration of PP1 in the membranes increased 40% between 2 and 14 days and a further 60% between 14 days and adult, whereas the PP1 enzyme activity in the membranes progressively decreased. In contrast to PP1, the protein concentration of PP2A remained constant in all fractions during post-hatch development, and the enzyme activity of PP2A did not change except for a decrease in the membrane-bound activity between 2 and 14 days. These results show that the subcellular distribution and activity of PP1 is selectively regulated during post-hatch development and that membrane association and inactivation of PP1 are independent events.

Tyrosine hydroxylase phosphorylation in digitonin-permeabilized bovine adrenal chromaffin cells: the effect of protein kinase and phosphatase inhibitors on Ser19 and Ser40 phosphorylation.

The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [gamma-32P]ATP, in the presence or absence of 10 microM Ca2+, 1 microM cyclic AMP, 1 microM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273-302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5-22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19-31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.

Protein phosphorylation and calcium uptake into rat forebrain synaptosomes: modulation by the sigma ligand, 1,3-ditolylguanidine.

The sigma ligand 1,3-di-O-tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca2+ concentration ([Ca2+]i) and blocked the depolarization-dependent increases in [Ca2+]i. These effects were inhibited by the sigma antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca2+]i and the KCI-evoked rise in [Ca2+]i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca2+]i was not mediated via downstream regulation of cyclic GMP levels. DTG increased 45Ca2+ uptake and efflux under basal conditions and inhibited the 45Ca2+ uptake induced by depolarization with KCI. The KCI-evoked rise in [Ca2+]i was inhibited by omega-conotoxin (omega-CgTx)-GVIA and -MVIIC but not nifedipine and omega-agatoxin-IVA. The effect of DTG on decreasing the KCI-evoked rise in [Ca2+]i was additive with omega-CgTx-MVIIC but not with omega-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca2+ levels via inhibition of N-type Ca2+ channels.

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Phosphorylation of synapsin I and dynamin in rat forebrain synaptosomes: modulation by sigma (sigma) ligands.

The effects of sigma (sigma) ligands on protein phosphorylation were examined in crude, rat forebrain synaptosomes. Synaptosomes were prelabelled with 32P(i) and incubated with the sigma ligands 1,3-di-o-tolylguanidine (DTG), (+)pentazocine and (-)pentazocine (3, 10, 30, 100, 300 microM), or haloperidol, reduced haloperidol, and (+)SKF 10,047 (100 microM). Aliquots were then incubated for 10 s in control (5 mM K+) or depolarising buffer (41 mM K+). All the sigma ligands increased basal phosphorylation of synapsin Ib and other proteins including dynamin, and inhibited the depolarisation-dependent increase in phosphorylation of synapsin Ib in synaptosomes. The effects of these ligands are not directly on protein kinases or protein phosphatases. This indicates that the sigma ligands are mediating their effects via interaction with sigma binding sites, and suggest, for the first time, that protein phosphorylation may be one mechanism through which sigma ligands produce their biological effects.

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of intracellular Ca2+ in the histamine H1 receptor-stimulated phosphorylation of Ser8, Ser19, Ser31, and Ser40.

Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+. In contrast, the H1-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 microM) and significantly reduced by prior exposure to caffeine (10 mM for 10 min) to deplete intracellular Ca2+. Tryptic-phosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31. In contrast, although a brief stimulation with nicotine (30 microM for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.

A fibroblast elongation factor purified from colon carcinoma cells shares sequence identity with TIMP-1.

We have previously reported that human colon cancer cells secrete a factor(s) which induces elongation of colon fibroblasts in vitro. Isolation of this factor led to the identification of a 55kD protein with fibroblast stretching activity. Two internal amino acid sequences identified in this protein (YEI; GFQALGDAADI) share complete homology with tissue inhibitor of metalloproteinase (TIMP-1).

Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization.

Dynamin I is a nerve terminal phosphoprotein with intrinsic guanosine triphosphatase (GTPase) activity that is required for endocytosis. Upon depolarization and synaptic vesicle recycling, dynamin I undergoes a rapid dephosphorylation. Dynamin I was found to be a specific high-affinity substrate for calcineurin in vitro. At low concentrations, calcineurin dephosphorylated dynamin I that had been phosphorylated by protein kinase C. The dephosphorylation inhibited dynamin I GTPase activity in vitro and after depolarization of nerve terminals. The effect in nerve terminals was prevented by the calcineurin inhibitor cyclosporin A. This suggests that in nerve terminals, calcineurin serves as a Ca(2+)-sensitive switch for depolarization-evoked synaptic vesicle recycling.

Differential activities of protein phosphatase types 1 and 2A in cytosolic and particulate fractions from rat forebrain.

The activities and concentrations of protein phosphatase type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.

Synaptosomal amino acid release: effect of inhibiting protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the role of protein phosphatases in regulating the release of amino acids from synaptosomes. Okadaic acid increased the basal release of the amino acids glutamate, aspartate and GABA. The effect was specific in that taurine was not released by either KCl or okadaic acid and there was no synaptosomal lysis or change in ATP/ADP ratios in the presence of okadaic acid. The okadaic acid-stimulated release of amino acids was, however, only a small proportion of that produced by KCl depolarisation. Since okadaic acid raised synaptosomal protein phosphorylation levels to those equivalent to that produced by KCl depolarisation, it is unlikely therefore that there is a direct causal relationship between protein phosphorylation and the release of amino acids. Nevertheless, that release of amino acids from synaptosomes can be elevated under basal conditions by okadaic acid treatment does suggest that okadaic acid-sensitive protein phosphatases have a modulatory role in this process.

Protein phosphatase activity in cyanobacteria: consequences for microcystin toxicity analysis.

Hepatotoxic microcystin levels in cyanobacteria (blue-green algae) were assessed by an assay based on inhibition of protein phosphatases type 1 (PP1) and type 2A (PP2A) in crude chicken forebrain extracts using 32P-labelled glycogen phosphorylase as substrate. While cyanobacteria are reported to be devoid of phosphorylase phosphatase activity, two samples obtained from cyanobacterial scums, containing predominantly Anabaena circinalis, were found to contain high levels of a phosphorylase phosphatase activity which completely masked the presence of microcystin. Furthermore, samples containing predominantly Microcystis aeruginosa but increasing Anabaena circinalis contained sufficient phosphorylase phosphatase activity to cause a fourfold underestimation of microcystin levels. Thus, protocols for microcystin toxicity analysis should take into account the possible presence of endogenous phosphatase activity, thereby preventing underestimation of toxin levels.

Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the protein phosphatases involved in the endogenous dephosphorylation of proteins in intact synaptosomes. Despite the fact that the calcium-dependent protein phosphatase (calcineurin) is most concentrated in synaptosomes and accounts for approximately 0.3% of synaptoplasmic protein, the majority of the dephosphorylation activity under both basal and depolarisation conditions is due to protein phosphatase type 1 (PP1) and/or protein phosphatase type 2A (PP2A). Nevertheless our results do suggest that calcineurin is active in synaptosomes and has 2 effects: a rapid, direct dephosphorylation of a limited range of substrates and an indirect activation of PP1 presumably by dephosphorylation of protein phosphatase 1 inhibitor-1.

The regulation and function of protein phosphatases in the brain.

Data emerging from a number of different systems indicate that protein phosphatases are highly regulated and potentially responsive to changes in the levels of intracellular second messengers produced by extracellular stimulation. They may therefore be involved in the regulation of many cell functions. The protein phosphatases in the nervous system have not been well studied. However, a number of neuronal-specific regulators (such as DARPP-32 and G-substrate) exist, and brain protein phosphatases appear to have particularly low specific activity, suggesting that neuronal protein phosphatases possess considerable and unique potential for regulation. Several early events following depolarization or receptor activation appear to involve specific dephosphorylations, indicating that regulation of protein phosphatase activity is important for the control of many neuronal functions. This article reviews the current literature concerning the identification, regulation, and function of serine/threonine protein phosphatases in the brain, with particular emphasis on the regulation of the major protein phosphatases, PP1 and PP2A, and their potential roles in modulating neurotransmitter release and postsynaptic responses.