Susan E. Wilson

Gap-junction disassembly and connexin 43 dephosphorylation induced by 18β-glycyrrhetinic acid

Gap‐junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor‐promoting chemicals have been identified that inhibit cell‐cell gap‐junctional coupling. Here, we provide morphological evidence that 18β‐glycyrrhetinic acid (18β‐GA), a saponin isolated from licorice root that is an inhibitor of gap‐junctional communication, caused the disassembly of gap‐junction plaques in WB‐F344 rat liver epithelial cells. This effect was dose (5–40 μM) and time dependent (1–4 h treatment). Gap‐junction channels in WB‐F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43‐P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18β‐GA was correlated with decreases in Cx43‐P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18β‐GA‐induced dephosphorylation of Cx43‐P2 and not its degradation or the inhibition of Cx43‐NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18β‐GA causes type 1 or type 2A protein phosphatase‐mediated Cx43 dephosphorylation coincident with the disassembly of gap‐junction plaques.

Retention of the Alzheimer's Amyloid Precursor Fragment C99 in the Endoplasmic Reticulum Prevents Formation of Amyloid β-Peptide

γ-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimers β-amyloid precursor protein (APP), resulting in the release of amyloid β-peptide (Aβ). The molecular identity of γ-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that γ-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Aβ. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimers disease resulted in a characteristic elevation of the Aβ42/Aβ40 ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of γ-secretase cleavage. Biogenesis of both Aβ40 and Aβ42 was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for γ-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as γ-secretase. The results suggest that presenilins may acquire the characteristics of γ-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.

A latent form of protein phosphatase 1 α associated with bovine heart myofibrils

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37 000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37 000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominately the α isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co2+ or Mn2+ alone but not by Ca2+, Mg2+, Ni2+, Cu2+ or Zn2+. Activation of the enzyme was not reversed by removal of Co2+, but Mn2+-activated phosphatase activity was partially reversed when Mn2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co2+. Since phosphatase 1α is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.

Isolation of a heat-stable protein activator of phosphorylase phosphatase

Phosphorylase phosphatase activity of protein phosphatase-1 [1] is believed to be regulated by: (i) Specific interaction of ligands with phosphorylase a; i.e.i by substrate directed-effects; (ii) Competition between phosphorylase and various other phosphoprotein substrates; (iii) Interaction of the phosphatase with heatstable inhibitors [2]. Two heat-stable inhibitors have been isolated from skeletal muscle, inhibitor-1 and inhibitor-2 [3-5]. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form [3,4]. The degree of phosphorylation of inhibitor-1 is under hormonal control [ 1 ]. While studying the heat-stable protein inhibitors of phosphorylase phosphatase from kidney, we have found an activator of phosphorylase phosphatase. To our knowledge such an activator has not been described. We have purified the heatstable protein activator from swine renal cortex 300-fold and have studied some of its properties. The activator stimulates phosphorylase phosphatase activity primarily by decreasing the Km of the enzyme for phosphorylase a.

Evidence for a latent form of protein phosphatase 1 associated with cardiac myofibrils

Detergent-purified myofibrils from bovine heart contained very little spontaneously active protein phosphatase 1 activity. Phosphatase 1, extracted from the myofibrils by freeze-thawing in the presence of 500 mM KCl, was markedly activated by cobalt/trypsin treatment. Myofibril phosphatase 1 was separated from phosphatase 2A by chromatography on heparin-Sepharose. The phosphatase 1 was isolated in a latent form. Pretreatment with trypsin released free catalytic subunit and increased activity about 25-fold. Addition of cobalt with the trypsin increased activity another 2-fold. The latent myofibril phosphatase 1 did not appear to be the same as previously characterized forms of protein phosphatase 1. We suggest that cardiac myofibril phosphatase 1 contains a unique inhibitory subunit which directs the enzyme to the myofibril and regulates the dephosphorylation of myofibril phosphoproteins.

Evidence that the heat-stable protein activator of phosphorylase phosphatase is histone H1

The heat-stable protein activator of phosphorylase phosphatase [FEBS Lett. 146, 331-334 (1982)] was located in the nuclear fraction. The activator was isolated from the nuclear fraction of various tissues. The largest amount was obtained from calf thymus and swine renal cortex. The activator protein from swine renal cortex was purified to apparent homogeneity. The activator had the same mobility as authentic histone H1 on sodium dodecyl sulfate or acetic acid/urea polyacrylamide gel electrophoresis. Phosphorylase phosphatase was activated by histone H1 or purified activator in an identical manner. Other histones were without effect or inhibited phosphorylase phosphatase activity. It is concluded that the heat-stable protein activator of phosphorylase phosphatase is histone H1.

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex.

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).

Effect of Inhibitors of Neuronal and Extraneuronal Uptake on the Accumulation and Metabolism of 3H-l-Norepinephrine in Rabbit Aorta

In the isolated adventitia of rabbit aorta, blockade of neuronal uptake decreased the accumulation and deamination of 3H-l-norepinephrine (3H-l-NE) whereas blockade of extraneuronal uptake decreased O-methylation. Thus most of the O-methylation occurs in the extraneuronal elements. In the isolated media, blockade of extraneuronal uptake decreased metabolism but not accumulation of 3H-l-NE. If NE metabolism was prevented by pretreatment with pargyline and U-0521, 3H-l-NE accumulation in the isolated media increased markedly and now inhibitors of extraneuronal uptake could decrease this accumulation of 3H-l-NE by the isolated media. Thus when MAO and COMT are intact, NE does not accumulate in the extraneuronal compartment containing these enzymes, probably because it is metabolized as fast as it enters the compartment. Since the level of 3H-l-NE in the isolated media exceeded the sorbitol space when MAO and COMT are intact, some 3H-l-NE must have accumulated in a compartment which does not contain these enzymes.