Phosphoprotein phosphatase activity of Novikoff hepatoma nucleoli.

Nucleoli from Novikoff hepatoma ascites cells contain phosphatase activity that acts upon ³²P-labeled nucleolar protein substrates. The activity is optimal near pH 7.0 and is inhibited by increasing concentrations of NaCl. The divalent cations CaCl₂, MnCl₂ and CoCl₂ at 6 mM inhibited phosphatase activity from 30–60%. ZnCl₂ completely inhibited the activity above 2 mM while EDTA and MgCl₂ had little effect. The activity was stimulated by dithiothreitol and inhibited by N-ethylmaleimide indicating a requirement for free sulfhydryl groups.     

Characterization of a phosphoprotein phosphatase activity in ribonucleoprotein particles from HeLa cell nuclei.

Ribonucleoprotein particles containing heterogenous nuclear RNA (hnRNP) have a phosphoprotein phosphatase activity. This activity is optimal at pH 7.5, inhibited by divalent cations and by increasing ionic strength above 200 mM NaCl, stimulated by 2-mercaptoethanol and inhibited by N-ethylmaleimide. It is clearly distinct from non specific alkaline phosphatase and resembles the phosphoprotein phosphatase present in Novikoff hepatoma nucleoli (Olson et al. B.B.R.C. (1976), 70, 717–721). This enzyme may be involved in regulating the phosphorylation level of hnRNP proteins in combination with the protein kinase previously described (Blanchard et al. Eur. J. Biochem. (1977) in press).     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells

Summary The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.     

Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma.

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.     

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 β-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 (A_0.5 = 0.6 μM). 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 α- and β-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.     

Phosphorylation of purified Novikoff hepatoma topoisomerase I

The purified Novikoff hepatoma nuclear phosphoprotein with a molecular weight of 110 kdalton and pI 8.4, was found to be a type I topoisomerase. When isolated from 32P-labeled Novikoff ascites cells or incubated in vitro with protein kinase, phosphoserine was found to be its major phosphorylated amino acid. The enzymatic activity of topoisomerase I was altered by changes in phosphorylation. Its activity was increased by protein kinase and it was decreased by alkaline phosphatase.     

Amino acid sequence and sites of phosphorylation in a highly acidic region of nucleolar nonhistone protein C23.

Novikoff hepatoma nucleolar nonhistone proteins, C23 and B23, contain highly acidic phosphorylated regions (Mamrack, M. D., et al. (1977) Biochem. Biophys. Res. Commun. 76, 150--157). Tryptic peptides from protein C23 containing these regions were purified by DEAE-Sephadex columns and paper electrophoresis at pH 1.8. One of these, peptide C23-Ca, was sequenced by combined automated and conventional methods. The proposed amino acid sequence is shown in eq 1. This peptide was found in three 32P-labeled forms with phosphoryl groups at positions 8 and 25, and probably 28. The highly acidic sequences adjacent to the phosphorylation sites represent a unique class of phosphorylation sites different from those in histones or substrates for cytoplasmic cAMP-dependent kinases. Ala-Ala-Pro-Ala-A5la-Pro-Ala-Ser-Glu-A10sp-Glu-Asp-Glu-Glu-A15sp-Asp-Asp-Asp-Glu-A20sp-Asp-Asp-Asp-Asp-S25er-Gln-Glu-Ser-Glu-G30lu-Glu-Asp-Glu-Glu-V35al-Met-Glu-Ile-Thr-P40ro-Ala-Lys (1).     

Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 mumol/mg of protein/min, with a Km of 4.7 microM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 microM), sodium vanadate (IC50 of 150 microM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 microM ZnCl or 10 mM NaF. Poly-(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.     

Nuclear matrix, hnRNA, and snRNA in friend erythroleukemia nuclei depleted of chromatin by low ionic strength EDTA

A novel and rapid procedure is described for the preparation of chromatin-depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60-80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50-100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus-specific phospho-protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalent cations.     

Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells

The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control.     

DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins

Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [³²P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.     

Association of protein C23 with rapidly labeled nucleolar RNA

The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [3H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [3H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA.(ABSTRACT TRUNCATED AT 250 WORDS)