Identification and preliminary characterization of two related proliferation-associated nuclear phosphoproteins.
Two nuclear phosphoproteins, pp35 and pp32, were purified from A20 cells, a murine B-lymphoblastoid cell line. Initially detected by cross-reactivity with antibodies to human erythrocyte protein 4.1, the 35- and 32-kDa proteins were purified by sequential fractionation of non-ionic detergent cell lysates on DEAE-cellulose, high performance liquid chromatography (HPLC)-anion-exchange chromatography, and HPLC hydroxylapatite chromatography. By two-dimensional peptide mapping, pp35 and pp32 are related but do not appear to represent sequential proteolytic products. Both pp35 and pp32 appear to be associated with cell proliferation. Antibodies specific for pp35 and pp32 show prominent intranuclear staining in A20 cells but only focal staining in normal murine lymphoid tissues. Quantitative immunoblotting showed that both pp35 and pp32 are, respectively, expressed at 5.9 x 10(4) and 7.0 x 10(4) copies/cell in small, dense resting B lymphocytes, increasing approximately 12- and 7-fold after polyclonal stimulation with lipopolysaccharide. When normalized to total cell protein, this represents specific inductions of approximately 4- and 2-fold. Expression of both pp35 and pp32 is constitutively high in populations of neoplastic B cell lines; moreover, both are expressed in the nuclei of intestinal crypt epithelial cells but not in other epithelial compartments in the same sections, suggesting that forms of pp35 and pp32 may be expressed in additional tissues and associated with proliferation.
Malek
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Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Katumuluwa
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Pasternack
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GR
D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D015180:Electrophoresis, Gel, Two-Dimensional; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D010750:Phosphoproteins; D002918:Chymotrypsin