ARTICLE |
Correspondence to: Tatsuji Haneji, Dept. of Histology and Oral Histology, School of Dentistry, University of Tokushima, 3-18-15, Kuramoto, Tokushima 770-8504, Japan. E-mail: tat-hane@dent.tokushima-u.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1
and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1
and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1
antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1
and nucleolin was changed. Expression of PP1
was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1
was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1
. (J Histochem Cytochem 50:11871193, 2002)
Key Words: protein phosphatase, nucleolin, osteoblast
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PROTEIN DEPHOSPHORYLATION, in cooperation with protein phosphorylation, plays roles in the regulation of cell metabolism, cell cycle progression, and cell differentiation (, PP1
, PP1
1, and PP1
2 (
The expression of PP1s was previously investigated at the mRNA and protein levels in various tissues and organs (1 (
2 (
Nucleolin has a molecular mass of 100110 kD and is an abundantly expressed phosphoprotein located mainly in the fibrillar components of nucleoli, where it associates with nascent preribosomal RNA. Nucleolin is involved in the regulation of ribosome biogenesis, cell proliferation and growth, embryogenesis, cytokinesis, and nucleogenesis ( (
and is a candidate substrate for this enzyme.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Alpha-modified Eagle's minimal essential medium was purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was obtained from JRH Biosciences (Lenexa, KS). Plastic dishes were from Falcon Plastics (Los Angeles, CA). Anti-PP1 antiserum was a gift from Drs. M. Nagao and H. Shima and was characterized previously (
Cell Culture
Human osteoblastic cell lines MG63 (-MEM containing 10% (v/v) FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and were maintained at 37C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every 3 days and subculturing was performed every 6 days by treatment of the cells with 0.25% trypsin together with 1 mM EDTA in Ca2+-, Mg2+-free PBS. For experiments, subconfluent cells were treated with the agents by adding small volumes of stock solution to the medium and culturing for appropriate periods. Cell modification was monitored by an Olympus IMT-2 phase-contrast microscope. For immunocytochemistry, the cells were plated on 18-mm round coverslips in 60-mm plastic dishes.
Cell Fractionation, SDS-PAGE, and Western Blotting
Cells cultured in 90-mm plastic dishes were washed twice with PBS, scraped into PBS, pelleted at 3000 x g, and resuspended in hypotonic buffer [20 mM Hepes (pH 7.2), 10 mM KCl, 1 mM MgC12, 1 mM DTT, 0.5 mM EDTA, 50 mM NaF, and 1 mM Na3VO4]. The cells were allowed to swell for 10 min on ice before lysis by addition of 0.1% NP-40 and 100 mM potassium acetate. After 5 min on ice and vortexing, their nuclei were pelleted by centrifugation for 10 min at 8000 x g, resuspended in lysate buffer containing 1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 5 mM EGTA in PBS, and used as the nuclear fraction, whereas the supernatant was taken as the cytosolic fraction. The nucleolar and nucleoplasmic fractions were prepared from purified nuclei according to the method of antibody diluted 1:2000 or 0.5 µg/ml of anti-nucleolin antibody. The membranes were washed four times within 30 min in PBSTween on a rotary shaker at 2022C. The washed membranes were incubated in PBSTween containing horseradish perioxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG (diluted 1:10,000) for 1 hr at 2022C. The membranes were washed as described and the proteins recognized by the anitbodies were visualized by using an ECL detection kit (Amersham Pharmacia Biotech; Uppsala, Sweden) according to the manufacturer's directions.
Immunoprecipitation
Cells cultured in 90-mm plastic dishes were washed twice with PBS, scraped into PBS, pelleted at 3000 x g, and resuspended in 500 µl of lysis buffer [150 mM Nacl, 1.0% NP-40, 50 mM Tris-HCl (pH 8.0), 50 mM NaF, and 1 mM Na3 VO4]. The lysate was preincubated for 30 min with 20 µl of protein A/G PLUS agarose (Santa Cruz Biotechnology; Santa Cruz, CA) and then incubated with 2 µl of anti-PP1 antiserum or 1 µl of anti-nucleolin antibody conjugated to 10 µl of protein A/G PLUS agarose. The reaction mixture was incubated for 6 hr at 4C. The immunocomplexes were washed four times with lysis buffer and resuspended in 40 µl of SDS electrophoresis sample buffer. The samples were boiled for 3 min and analyzed by SDS-PAGE and Western blotting using the anti-nucleolin or anti-PP1
antibody.
Immunocytochemistry
The cells on the coverslips were washed three times with PBS and fixed with 3.7% formaldehyde for 10 min at 2022C, followed by methanol permeabilization for an additional 20 min at -20C. Nonspecific binding sites were blocked with 4% BSA in PBS for 10 min at 2022C in a humidified atmosphere. Having been rinsed with cold PBS, the coverslips were incubated simultaneously with anti-PP1 antibody diluted 1:200 and 5 µg/ml of the IgG fraction of anti-nucleolin antibody in 4% BSA for 45 min at 2022C. After three washes with 0.1% BSA in PBS containing 0.05% Tween-20 (PBSTween) over a 15 min period at 2022C, the cells were incubated with a mixture of tetramethylrhodamine isothiocyanate (TRITC, rhodamine)-conjugated sheep anti-rabbit IgG (Chemicon International; Temecula, CA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel-Organon Teknika; Turnhout, Belgium), both diluted 1:300 in 4% BSA in PBS for another 45 min at 2022C. The coverslips were washed as described above and mounted while wet with PermaFluor aqueous mounting medium (Lipshow; Pittsburgh, PA). The samples were examined under an Olympus BX50 microscope equipped with epifluorescence illumination (BX-FLA) with a U-MWIG filter for rhodamine and a U-MNIBA filter for FITC. The U-MNIBA filter separates FITC from rhodamine or Texas Red. The staining reaction was not observed when FITC-labeled cells were examined with a filter for rhodamine (a U-MWIG filter). Rhodamine-labeled cells were not detected with a filter for FITC (a U-MNIBA filter). Photomicrographs were taken on Fuji Sensia 400 film using the automatic exposure (PM-30).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of PP1 and Nucleolin in MG63 Cells
Fig 1 shows that the anti-PP1 antibody interacted with a band corresponding to the 37-kD protein present in the nuclear and nucleolar fractions prepared from MG63 cells. However, very few immunoreactive proteins were detected in the cytosolic fraction. The anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole cell lystate. The antibody recognized the 100-kD protein in nuclear and nucleolar fractions, and the staining intensity was stronger in the latter. Normal rabbit serum and normal mouse serum did not recognize any proteins prepared from cultured MG63 cells (data not shown).
|
Co-localization of PP1 and Nucleolin in MG63 and Saos-2 Cells
In previous reports, we and others demonstrated that PP1 localized in the nucleolus in mouse osteoblastic MC3T3-E1 cells and Swiss 3T3 cells (
and nucleolin would be localized in the same site in the nucleolus, we fixed, permeabilized, and stained cultured MG63 and Saos-2 cells with the anti-PP1
polyclonal antibody and the anti-nucleolin monoclonal antibody. Fig 2 shows the distribution of PP1
and nucleolin in MG63 and Saos-2 cells. With the anti-PP1
antibody, nucleolus-like bodies were intensely stained and visible as red fluorescence due to the rhodamine-conjugated second antibody (Fig 2a). A monoclonal anti-nucleolin antibody stained the same sites of the PP1
-positive regions, which gave a green fluorescence because an FITC-conjugated second antibody was used (Fig 2b). The merged view confirmed that PP1
and nucleolin were localized in the same sites in MG63 cells because the reaction was visible as yellow fluorescence (Fig 2c). Fig 2 also shows that the anti-PP1
antibody and the anti-nucleolin antibody stained the nucleolus-like materials in Saos-2 cells (Fig 2d and Fig 2e). The positive sites were stained as yellow fluorescence in the merged view, indicating that PP1
and nucleolin were also localized in the same sites in Saos-2 cells (Fig 2f). The staining reaction was not observed when normal rabbit serum or normal mouse serum was used as a primary antibody in both MG63 cells and Saos-2 cells (data not shown).
|
PP1 Associates with Nucleolin
To determine whether PP1 could associate with nucleolin, the whole cell lysate prepared from cultured MG63 cells was immunoprecipitated with the anti- PP1
antibody or with the anti-nucleolin antibody. The immunoprecipitants were analyzed by Western blotting using the anti-nucleolin antibody (Fig 3a) or the anti-PP1
antibody (Fig 3b). The anti-nucleolin antibody interacted with a protein precipitated with the anti-PP1
antibody. However, this antibody did not recognize any proteins precipitated with normal rabbit serum (Fig 3a). The molecular weight of the interactive protein was estimated as 100 kD, corresponding to the molecular weight of nucleolin. The anti-PP1
antibody interacted with a protein precipitated with the anti-nucleolin antibody. The interacted protein was estimated as 37 kD, corresponding to PP1
. The anti-PP1
antibody did not interact with the proteins precipitated with normal mouse serum (Fig 3b). As positive controls, the results for whole cell lysate stained with the anti-nucleolin antibody or anti-PP1
antibody are also shown in Fig 3a and Fig 3b, respectively.
|
Alteration of PP1 and Nucleolin in Actinomycin D-treated MG63 Cells
The changes of PP1 and nucleolin expression in the actinomycin D-treated MG63 cells were examined by histochemical and Western blotting procedures. With the PP1
antibody, the positive bodies became smaller, increased in number, and were distributed over the nuclei instead of being located in nucleoli (Fig 4a). The anti-nucleolin antibody also recognized the same bodies as nucleolin-containing materials and was distributed all over the nuclei (Fig 4b). The merged view confirmed that PP1
and nucleolin were distributed in the same bodies in the nuclei, as these bodies appeared as yellow fluorescence (Fig 4c). Fig 5 shows the results obtained from Western blotting analysis for nucleolin and PP1
in the actinomycin D-treated cells. The amount of 100-kD nucleolin did not change in the actinomycin D-treated cells. However, the intensity of the 95-kD band increased in the actinomycin D-treated cells in a dose-dependent manner up to 10 nM (Fig 5, upper panel). The staining intensity of PP1
in the actinomycin D-treated cells also increased in a dose-dependent manner (Fig 5, lower panel).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The specific localization of the catalytic subunit of PP1 in nucleoli in MG63 cells and Saos-2 cells indicates that the nucleolus is the site at which this enzyme functions. These findings confirm our previous results (
1 rather than PP1
was localized within the nucleolus (
is quite interesting. It should be noted that PP1
does not exhibit any evidence of nuclear localization signals in its sequence (
have been shown to freely diffuse into the nucleus without the necessity for specific translocation sequences. The non-catalytic subunits of PP1s have a targeting function, enabling the phosphatases to associate with a particular cellular fraction. Once in the nuclei, PP1s bind to immobile components to maintain their nuclear localization. The highest concentration of PP1s is found in the nucleus, where the enzymes are both in the nucleoplasm and associated with heterochromatin (
, is not known at present.
We paid much attention to the relationship between PP1 and nucleolin in the present study. We demonstrated that PP1
was localized in the nucleolus in cultured MG63 and Saos-2 cells and that this enzyme was associated with nucleolin. The function of PP1s depends on the localization of these enzymes in cells. Because, like protein kinases, protein phosphatases should be associated with or located near to their substrates (
should be present in the nucleolus. PP1 binding proteins are believed to have an R/KV/IXF, R/KXV/IF (
and nucleolin should be indirect, and some regulatory subunits might exist that allow PP1
to bind to nucleolin. However there remains the possibility that nucleolins bind directly to PP1 by unknown binding domains or mechanisms. The interaction between PP1
and nucleolin demonstrated by immunoprecipitation and Western blotting analysis suggests that PP1
might be the enzyme that dephosphorylates nucleolin. Although whether a 95-kD protein is a dephosphorylated form of nucleolin is not clear at present, the amount of a 95-kD protein decreased after calyculin A treatment in MG63 cells (unpublished data). These findings suggest that nucleolin could be partially dephosphorylated into a 95-kD protein detected with an anti-nucleolin antibody. Another possibility, that nucleolin is partially proteolyzed into a 95-kD protein, is not ruled out. It was also reported that localization of PP1
was similar to that of nucleophosmin and that nucleophosmin could be a candidate substrate for PP1
(
Interestingly, the 95-kD protein was present in the nucleoplasm and cytoplasm in MG63 cells. Once nucleolin is dephosphorylated, it might be translocated from the nucleolus to the nucleoplasm or cytoplasm and remain there by some mechanism yet to be examined. The relocation mechanism might involve the reversible phosphorylation of the nucleolin in the cytoplasm by cell cycle-dependent kinases. It was reported that nucleolin is phosphorylated by several kinases, including casein kinase, p34cdc2 kinase, and protein kinase C ( is reported to be associated with Rb protein (
may be the enzyme that dephosphorylates Rb proteins (
Actinomycin D, an anti-tumor antibiotic, is known to inhibit RNA synthesis. When cultured mammalian cells are exposed to relatively low concentrations of actinomycin D, the synthesis of the 45S nucleolar precursor of 1828s rRNA is inhibited, whereas there is little or no effect on the synthesis of tRNA and 5S rRNA ( and nucleolin redistributed from the nucleoli to the nucleoplasm after treatment with 10 nM actinomycin D for 4 hr. This condition is enough to cause inhibition of rRNA rather than mRNA synthesis and causes translocation of nucleophosmin (B23) from the nucleolus to the nucleoplasm in HeLa cells (
and 95-kD nucleolin are regulated by the action of kinases and phosphatases. Although the mechanisms of PP1
expression and the role of 95-kD nucleolin are not known, the interaction between PP1
and nucleolin may be correlated with dephosphorylation of nucleolin in MG63 cells. The idea that the function of nucleolin is coupled with the phosphorylation status is supported by the observation that active rRNA transcription is correlated with the phosphorylation level of nucleolin (
![]() |
Footnotes |
---|
1 Present address: Dept. of Anatomy, School of Dentistry, University of Tokushima, Tokushima 770-8504, Japan.
![]() |
Acknowledgments |
---|
Supported in part by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (HM) and by a grant from the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (TH).
We thank Ms Eiko Sasaki for skillful technical assistance.
Received for publication December 10, 2001; accepted March 27, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andreassen PR, Lacroix FB, VillaMoruzzi E, Margolis RL (1998) Differential subcellular localization of protein phosphatase-1,
1, and
isoforms during both interphase and mitosis in mammalian cells. J Cell Biol 141:1207-1215
Belenguer P, Baldin V, Mathieu C, Prats H, Bensaid M, Bouche G, Amalric F (1989) Protein kinase NII and the regulation rDNA transcription in mammalian cells. Nucleic Acids Res 17:6625-6635[Abstract]
Belenguer P, CaizerguesFerrer M, Labbe JC, Doree M, Amalric F (1990) Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase. Mol Cell Biol 10:3607-3618[Medline]
Borer RA, Lehner CF, Eppenberger HM, Nigg EA (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56:379-390[Medline]
CaizerguesFerrer M, Belenguer P, Lapeyre B, Amalric F, Wallace MO, Olson MOJ (1987) Phosphorylation of nucleolin by a nucleolar type NII protein kinase. Biochemistry 26:7876-7883[Medline]
Derenzini M (2000) The AgNORs. Micron 31:117-120[Medline]
Egloff MP, Johnson DF, Moorhead G, Cohen PT, Cohen P, Barford D (1997) Structural basis of the recognition of regulatory subunits by protein phosphatase 1. EMBO J 16:1876-1887
Erard MS, Belenguer P, CaizerguesFerrer M, Pantaloni A, Amalric F (1988) A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1. Eur J Biochem 175:525-530[Abstract]
Franceschi RT, James WM, Zerlauth G (1985) 1, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in human osteosarcoma cell line. J Cell Physiol 123:401-409[Medline]
Ginisty H, Sicard H, Roger B, Bouvet P (1999) Structure and functions of nucleolin. J Cell Sci 112:761-772
Haneji T, Morimoto Y, Shirakawa S, Kobayashi S, Kaneda C, Shima H et al. (1998) Subcellular localization of protein phosphatase type 1 isotypes in mouse osteoblastic cells. Biochem Biophys Res Commun 248:39-43[Medline]
Hashikawa T, Nakazawa K, Mikawa S, Shima H, Nagao M (1993) Immunohistochemical localization of protein phosphatase isoforms in the rat cerebellum. Neurosci Res 22:133-136
Huang HB, Horiuchi A, Watanabe R, Shih SR, Tsay HJ, Li HC, Greengard P et al. (1999) Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2. J Biol Chem 274:7870-7878
Hunter T (1995) Protein kinase and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225-236[Medline]
Inagaki N, Ito M, Nakano T, Inagaki M (1994) Spatiotemporal distribution of protein kinase and phosphatase activities. Trens Biochem Sci 19:448-452
Jagiello I, Beullens M, Stalmans W, Bollen M (1995) Subunit structure and regulation of protein phosphatase-1 in rat liver nuclei. J Biol Chem 270:17257-17263
Kakinoki Y, Mizuno Y, Takizawa N, Imai Y, Miyazaki T, Kikuchi K (1994) TGFß1 suppresses EGF-induced increase in nuclear type 1 protein phosphatase activity at the G1/S transition of hepatocyte proliferation. FEBS Lett 352:356-360[Medline]
Kotani H, Ito M, Hamaguchi T, Ichikawa K, Nakano T, Shima H, Nagao M et al. (1998) The isoform of protein phosphatase type 1 is localized in nucleolus and dephosphorylates nucleolar phosphoproteins. Biochem Biophys Res Commun 249:292-296[Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]
Liu HT, Yung BYM (1999) In vivo interaction of nucleophosmin/B23 and protein C23 during cell cycle progression in HeLa cells. Lett 144:45-54
Muramatsu M, Hayashi T, Saki M, Takai K, Kashiyama T (1974) Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells. Exp Cell Res 88:245-251[Medline]
Murányi A, Erdodi F, Ito M, Gergely P, Hartshorne DJ (1998) Identification and localization of myosin phosphatase in human platelets. Biochem J 330:225-231[Medline]
Nelson DA, Kruncher NA, Ludlow JW (1997) High molecular weight protein phosphatase type 1 dephosphorylates the retinoblastoma protein. J Biol Chem 272:4528-4535
Norbury C, Nurse P (1992) Animal cell cycles and their control. Annu Rev Biochem 61:441-470[Medline]
Perry RP, Kelley DE (1970) Inhibition of RNA synthesis by actinomycin D: characteristic dose-responses of different RNA species. J Cell Physiol 76:127-140[Medline]
Puntoni F, VillaMoruzzi E (1997) Association of protein phosphatase-1 with the retinoblastoma protein and reversible phosphatase activation in mitotic HeLa cells and in cells released from mitosis. Biochem Biophys Res Commun 234:704-708
Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, Shull S et al. (1987) Characterization of human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 47:4961-4966[Abstract]
Sasaki K, Shima H, Kitagawa Y, Irino S, Sugimura T, Nagao M (1990) Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1 alpha gene in rat hepatocellular carcinomas. Jpn J Cancer Res 81:1272-1280[Medline]
Shima H, Haneji T, Hatano Y, Kasugai I, Sugimura T, Nagao M (1993a) Protein phosphatase 12 is associated with nuclei of meiotic cells in rat testis. Biochem Biophys Res Commun 194:930-937[Medline]
Shima H, Haneji T, Hatano Y, Nagao M (1993b) Expression of protein phosphatase 1 and 2A in rat testis. Adv Prot Phosphatase 7:489-499
Shima H, Hatano Y, Chun YS, Sugimura T, Zang Z, Lee EYC, Nagao M (1993c) Identification of PP1 catalytic subunit isotypes PP1 gamma 1, PP1 delta and PP1 alpha in various rat tissues. Biochem Biophys Res Commun 192:1289-1296[Medline]
Shirakawa S, Mochizuki H, Kobayashi S, Takehara T, Shima H, Nagao M, Haneji T (1996) Immunohistochemical and immunoblotting identification of protein phosphatase 11 in rat salivary glands. FEBS Lett 393:57-59[Medline]
Shirato H, Shima H, Sakashita G, Nakano T, Ito M, Lee EYC, Kikuchi K (2000) Identification and characterization of a novel inhibition of type 1 protein phosphatase. Biochemistry 39:13848-13855[Medline]
Schneider HR, Mieskes G, Issinger OG (1989) Specific dephosphorylation by phosphatase 1 and 2A of a nuclear protein structurally and immunologically related to nucleolin. Possible influence on the regulation of rRNA synthesis. Eur J Biochem 180:449-455[Abstract]
Srivastava M, Pollard HB (1999) Molecular dissection of nucleolin's role in growth and cell proliferation: new insights. FASEB J 13:1911-1922
Towbin H, Staehlin T, Gordon J (1979) Elctrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354[Abstract]
TrinkleMulcahy L, Sleeman JE, Lamond AI (2001) Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J Cell Sci 114:4219-4228
Ware S, Hemming BA (1995) Serine/threonine protein phosphatases. Biochem J 311:17-29[Medline]
Wu MH, Lam CY, Yung BYM (1995) Translocation of nucleophosmin from nucleoli to nucleoplasm requires ATP. Biochem J 305:987-992[Medline]
Yung BYM, Busch RK, Busch H, Mauger AB, Chan PK (1985) Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI5.1). Biochem Pharmacol 34:4059-4063[Medline]
Zhou G, Seibenhener ML, Wooten MW (1997) Nucleolin is a protein kinase C- substrate. Connections between cell surface signaling and nucleus in PC12 cells. J Biol Chem 272:31130-31137