Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23. Implication for the role of the nucleolus in early transduction of mitogenic signals.
We have previously described and characterized a nuclear protein at 40 kDa/pI 5 termed "numatrin" which is tightly bound to the nuclear matrix. We demonstrated that a rapid increase in the synthesis of numatrin at early G1 phase is closely correlated with receptor-mediated induction of cellular proliferation by various mitogens and that elevated amounts of numatrin are found in tumor cells, suggesting that numatrin may have an important role in regulation of cellular growth in normal and malignant cells. Further experiments were undertaken to compare the biochemical characteristics of numatrin to those of other known proteins that are associated with cellular mitogenesis. Comparison of the electrophoretic mobility of numatrin with the proliferation cell nuclear antigen/cyclin showed that these proteins are not identical. However, numatrin had an identical electrophoretic migration on two-dimensional gel electrophoresis to that of a previously described nucleolar protein B23. The tryptic digest peptide map of 125I-labeled B23 was identical to that of numatrin on two-dimensional thin layer electrophoresis/chromatography. Labeling of cells with 32P further showed that numatrin is a major phosphoprotein as previously reported for protein B23. Using the protocol for purification of B23, we purified numatrin from nucleoli of HL-60 cells and produced two polyclonal antibodies (303 and 339) to this protein. We further show that numatrin is recognized by anti-B23 monoclonal antibody as well as by polyclonal antibodies 303 and 339 in enzyme-linked immunosorbent assay. Conversely, these anti-numatrin polyclonal antibodies cross-react with protein B23 as shown in immunoblot analysis. These results, taken collectively, prove that numatrin is identical to the nucleolar protein B23 and thus suggest that protein B23 and events which occur at the nucleolus might have an important role in early transduction of mitogenic signals at the G1 phase of the cell cycle.
Feuerstein
N
N
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Chan
P K
PK
Mond
J J
JJ
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification*; D000090243:Nucleophosmin; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000906:Antibodies; D000936:Antigen-Antibody Complex; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D000090243:Nucleophosmin; D014357:Trypsin