Gottfried Mieskes

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (ICa) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100μM) produced an increase ofICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.ICa was increased from 11.3±0.8μA cm−2 to 19.0±1.1μA cm−2 (n=4) by 5μM-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the β-adrenergic increase ofICa was markedly prolonged by OA. The β-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onICa is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

On a possible role of fructose 2,6-bisphosphate in regulating photosynthetic metabolism in leaves

1. INTRODUCTION In photosynthesis two distinct FBPases are in- volved: a stromal enzyme participating in the Cal- vin cycle and a cytosolic enzyme involved in sucrose synthesis. Regulation of these enzymes is needed to permit a balanced withdrawal of carbon from the chloroplast stroma, provided by the Cal- vin cycle or by the degradation of assimilatory starch, for synthesis of sucrose and other carbon utilizing reactions in the cytosol. The stromal FBP- ase is known to be activated in the light by an in- terconversion catalysed by thioredoxin

The protein-specific phosphatase 1 antagonizes the β-adrenergic increase of the cardiac Ca current

In isolated ventricular cells from the adult guinea pig heart the slow Ca current was recorded during β-adrenergic stimulation and during cell dialysis with a protein-specific phosphatase-1 (PPase-1).The increase in the amplitude of ICa during bath application of isoprenaline (5×10−8M) could be completely reversed by dialysing the cell with 2 μM PPase-1. Lower enzyme concentrations produced smaller effects. The control amplitude of ICa was only little affected by dialysis with PPase-1.The result suggests 1) that PPase-1 is a likely candidate for the downregulation of Ca channels, 2) that Ca channels can open in the dephosphorylated state.

Molecular cloning and primary structure of a protein phosphatase 2C isoform

Complementary DNA encoding the isoform of protein phosphatase 2C, termed PP2C2, has been isolated. The cDNA predicts a protein of 390 amino acid residues with a molecular mass of 42,888 Da. The protein displays 76% identity to the PP2C1 isoform.

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP‐ and cGMP‐dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP‐dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.

Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis‐Golgi network

© 1997 Federation of European Biochemical Societies.

Protein phosphatase 2A reverses inhibiton of inward rectifying K+ currents by thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin‐releasing hormone (TRH) reduces an inwardly rectifying K+ current in whole‐cell voltage‐clamped GH3 rat anterior pituitary cells. The TRH effect depends on the maintenance of a background level of Ca2+ in the pipette buffer, and is rapidly minimized by the intracellular dialysis produced under whole‐cell conditions. Introduction of ADP‐NH‐P, a non‐hydrolizable ATP analog, in the pipettes, nearly abolishes the TRH‐evoked inhibition. The TRH‐induced reduction of the inwardly rectifying current is significantly enhanced by incubation of cells 2–4 h with cholera toxin, but not by inclusion of 1 mM cyclic AMP in the pipettes. Under control whole‐cell conditions, the reduction caused by TRH is not reversed upon washout of the neuropeptide. However, this effect is readily reversed by addition of purified catalytic subunits of protein phosphatase 2A (PP‐2Ac) but not PP‐1c to the buffer used to fill the patch pipettes. Among previous results with PP inhibitors, these data indicate that PP2A is involved in the phosphorylation/dephosphorylation mechanism(s) that regulate the delayed TRH effects on GH3 cell excitability.

A type 2A protein phosphatase dephosphorylates the elongation factor 2 and is stimulated by the phorbol ester TPA in mouse epidermis in vivo

Mouse epidermal cytosol contains a protein phosphatase with M r, 38000, which dephosphorylates the elongation factor 2 (EF‐2) of protein biosynthesis and is stimulated after topical application of TPA to mouse skin [(1988) Biochem. Biophys. Res. Commun. 153, 1129‐1135], Dephosphorylation of EF‐2 by this phosphatase is inhibited by okadaic acid at concentrations as low as 10−8 M, but not by heparin up to concentrations of 600 . The catalytic subunit of protein phosphatase 2A (PP2Ac) with EF‐2 as a substrate exhibits the same sensitivity towards okadaic acid and insensitivity towards heparin as the EF‐2 phosphatase of epidermal cytosol. The catalytic subunit of protein phosphatase 1 (PP1c) is strongly suppressed by heparin and less sensitive towards okadaic acid than PP2Ac. PP2Ac is around 50 times more efficient in dephosphorylating EF‐2 than PP1c. These data indicate that the TPA‐stimulated EF‐2 phosphatase in epidermal cytosol is a type 2A protein phosphatase.

A membrane-associated creatine kinase (EC 2.7.3.2) identified as an acidic species of the non-receptor, peripheral v-proteins in Torpedo acetylcholine receptor membranes

not received Function of one of the M, 43000 proteins Membrane-cusociated enzyme Acetyfcholine receptor-rich membrane Peripheral membrane protein Adenosine S ‘-triphosphate:creatine phosphotransferase (EC 2.7.3.2) A group of proteins named the 4%~protein [I ,2], the v-peptide [3], the v-doublet [4], or the iw, 43ooO proteins [S] is probably one of the most abundant non-receptor constituents of acetylcholine receptor (AChR)-rich membranes. Early studies associated the Y-proteins with the site of action of local anaesthetics [6], but it was subsequently shown that their extraction modified neither the binding of these ligands nor the AChRcontrolled ion permeability f2,7] + More recently, enhanced freedom of AChR rotational motion [S-IO] has been observed after extraction of these (and other) non-receptor peripheral proteins by alkaiine stripping. In [4] the involvement of the Yproteins in processes like synapse formation during ontogenesis, receptor clustering and stabilization of the adult synapse was proposed on the basis of the observed changes in AChR supramolecular organization which follow on from extraction of these proteins. Subsequently, the stabilization of one of the AChR oligomeric species, the 13 S dimer, was faund to be coincident with the lack of extraction of the Y-proteins [I I]. Antibodies raised against alkaline extracts of Torpedo membranes outline the profile of the mamm~i~n neuromuscular junction [12], and purified anti-pprotein antibodies reveal that their antigenic determinants are predominantly exposed on the inner, cytoplasmic face of the AChR-rich membrane [ 111. Lactoperaxidase iodination of holey vesicles confirms this localization [ 131. Aside from the heterogeneity apparent in conventional NaDodS04 electrophoresis [4,14], charge heterogeneity is displayed by the p-proteins [5,15]. The more basic subspecies, ~1, remains associated with AChR membranes after extensive purification by affinity partition, whereas the more acidic subspecies, YZ_ and ~3, are also found in dectrocyte soluble extracts [5]. The tightness of the association with the membrane is thus variabie for the different Y-proteins, which do not occur exclusively in membrane-bound form. Exogenous cross-linking of AChR membranes has recently been described as being related to the disappearance of PI, and appearance of a filamentous network [16], though neither the chemical nature of this network nor its relationship with ~1 were shown. Thus it does not necessarily follow that the Volume 152, number 2 FEBS LETTERS February 1983 modulation of AChR mobility depends on the presence of ~1 [16]. This work concerns the finding of creatine kinase (EC 2.7.3.2, ‘CK’), one of the phosphokinases that catalyzes phosphoryl group transfer to a guanidino function, in AChR-rich membranes from Torpedo electric tissue. Enrichment of this enzymatic activity coincides with the purification of the Y-proteins and more specifically, with the subspecies of intermediate pZ-value (6-6.5). Specific cross-reactivity of the AChR membrane-associated proteins with anti BB (brain) type of CK antibodies is observed. No such crossreactivity is apparent in AChR membraneassociated p-proteins with antibodies against the MM (muscle) type of isoenzyme. The kinetic properties of the membrane-associated and soluble (electric tissue) CK do not differ; the apparent Michaelis constants for MgADP and creatine phosphate are also similar to those found in the CK isoenzyme (MM type) present in Torpedo skeletal muscle. 2. MATERIALS AND METHODS 2.1. membrane purification and extraction procedures Native or ~-ethylmaleimide alkylated AChR membranes from Torpedo marmorata were isolated by the procedures reported in [ 17). Membrane fractions of relatively low spec. act. were chosen for most of the purification steps in order to maximize the contribution of non-receptor proteins. The immune blotting experiments were performed on both low and high spec. act. ([3H]~neurotoxin) membrane fractions. Alkaline extraction of non-receptor proteins was done essentially as in 12) with the modifications given in [ 11). Alternatively, lithium di-iodo-salicylate extraction of the membranes was carried out 17). 2.2. Thiol-Sepharose chromatography The methods usually applied for the purification of mammalian CK were attempted on alkaline or lithium diiodo-salicylate extracts of AChR membranes. Successive ethanol extractions (50-70%) (see [18]) were done at 0°C. After dialysis of the ethanol extract against 50 mM Tris-HCI buffer (pH 7.4) containing l-2 mM ,&-mercaptoethanol and concentration by vacuum dialysis, the extracts were chromatographed through p-hydroxymercuribenzoate-Agarose CL-BB, or Blue Sepharose CL-68 as described in 119) and [20], respectively. Both procedures have been successfully applied to CK purification, in particular for obtaining the labile fish enzymes [20]. 2.3. Gel electrophoresis and isoelectric focusing NaDodSOd-PAGE gel electrophoresis in slab systems followed the procedure in [21], except that samples were not heated for the reasons given in [4]. The 2-dimensional system in [22] was used without modifications. Integration of the Coomassie Blue-stained spots was done after digitaiisation of the data acquired with the aid of a high resolution vidicon camera. Computer processing of the digitised optical density profile provided: (a) a signal-averaged image of a rectangular frame of the stained gel after background subtraction; (b) integrated area measurements and average positions of the stained spots. 2.4. Immunological characterization of CK in AChR membranes Rabbit antisera against the MM and BB-CK isoenzymes were provided by Dr J.-C. Perriard, ETH (Zurich). Immune replicas of AChR membrane polypeptides were made on nitrocellulose paper as in 111). After saturation of unspecific sites with 5% horse serum in 50 mM Tris-HCl buffer (pH 7.4) for 2 h, 1: 100 dilutions of the first antibody in the same buffer were incubated overnight. Identification of the NaDodSOd-denatured antigens was made following reaction with a second (goat) peroxidase, FITC or rhodamine-labelled anti-rabbit IgG (Miles) also at a 1: 100 dilution. 2.4. determination of CK activity and enzyme kinetics The coupled enzyme assay: creatine-P + ADP & creatine + ATP glucose + ATP hexokinase glucose-6-P + ADP glucose-6-P + NADP+ GBP-DH gluconate-6-P +

Metabolite‐controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP‐dependent protein kinase

In hepatocytes 32P‐incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L‐alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one‐dimensional mapping on sodium dodecyl sulphate‐polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P‐incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP‐dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP‐dependent protein kinase but that the state of phosphorylation is modified by metabolite control.