Keith K. Schlender

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.

Activation of Protein Phosphatase 1 FORMATION OF A METALLOENZYME

The recombinant catalytic subunit of protein phosphatase 1 is produced as an inactive enzyme which can be activated by Mn2+ (Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M. F., and Lee, E. Y. C.(1992) J. Biol. Chem. 267, 1484-1490). In this report, we have investigated the effects of divalent cations on the activity of recombinant catalytic subunit of protein phosphatase 1. Latent phosphatase 1 can be activated by Co2+ or Mn2+, whereas other metal ions tested including Fe2+, Zn2+, Mg2+, Ca2+, Cu2+, or Ni2+ were not effective or were only weakly effective in activating the enzyme. The Mn2+-stimulated activity was susceptible to inactivation by EDTA; however, the Co2+-activated phosphatase was stable after dilution and chelation of the Co2+ with excess EDTA. After stable activation of phosphatase 1 using 57Co2+, a stoichiometric amount of 57Co2+ was shown to be tightly bound to phosphatase 1. These findings demonstrate for the first time the generation of a stable metalloenzyme form of phosphatase 1. Fe2+ reversibly deactivated the Co2+-stimulated activity, but did not displace the bound Co2+. Interestingly, treatment of the enzyme with a combination of Fe2+ and Zn2+ (but not the individual metal ions) significantly activated phosphatase 1. These results suggest that at least two metal binding sites exist on the enzyme and that protein phosphatase 1 may be an iron/zinc metalloprotein in vivo.

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle.

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.

Protein Phosphatases 1 and 2A Dephosphorylate B-50 in Presynaptic Plasma Membranes from Rat Brain

The protein B‐50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phospha‐tase type 1 and 2A (PP‐1 and PP‐2A)‐like activities. The present studies further demonstrate that B‐50 is dephosphorylated not only by a spontaneously active PP‐1‐like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 μg of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor‐2 and by high concentrations (μM) of okadaic acid, identifying it as a latent form of PP‐1. In the presence of inhibitor‐2 to block PP‐1, histone H1 (16–64 μg/ml) and spermine (2 mM) increased B‐50 dephosphorylation. This sensitivity to poly‐cations and the reversal of their effects on B‐50 dephosphorylation by 2 nM okadaic acid are indicative of PP‐2A‐like activity. PP‐1‐ and PP‐2A‐like activities from SPM were further displayed by using exogenous phosphorylase a and histone H1 as substrates. Both PP‐1 and PP‐2A in rat SPM were immunologically identified with monospecific antibodies against the C‐termini of catalytic subunits of rabbit skeletal muscle PP‐1 and PP‐2A. Okadaic acid‐induced alteration of B‐50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity‐purified anti‐B‐50 immunoglobulin G. These results provide further evidence that SPM‐bound PP‐1 and PP‐2A‐like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B‐50.

Histone H1-stimulated phosphorylase phosphatase from rabbit skeletal muscle.

A major rabbit skeletal muscle phosphorylase phosphatase activity which is markedly stimulated by histone H1 has been resolved from inhibitor-sensitive phosphorylase phosphatase (type-1 phosphatase), glycogen synthase kinase 3-activated phosphatase, phosphatase heat-stable inhibitor proteins, and alkaline phosphatase activity by various purification techniques. Evidence is presented that this phosphatase is a high-molecular weight form of a type-2 phosphatase. Our data suggest that this phosphatase may be regulated by histone H1, protamine or analogous polycationic compounds.

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.

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site‐specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N‐bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71–122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97‐Thr‐Gly‐Ala‐Ser‐Gly‐Ser(PO4)‐Phe‐Lys105, supports the contention that basic amino acid residues C‐terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.