Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts.
Jurkat T-lymphoblasts were induced to undergo apoptosis by treatment with either EGTA (5 mM/24 h) or a high concentration of lovastatin (100 microM/48 h) to identify proteins that exhibited coordinate regulation between the two treatments and thus provide candidate proteins in the common apoptotic induction pathway. A pure population of apoptotic cells, as determined by morphology, "DNA laddering," and flow cytometry, was obtained by Percoll density gradient centrifugation. Cells of increased buoyant density were clearly apoptotic by all criteria. Following this gradient centrifugation, the cells were labeled with [35S]methionine/cysteine, and lysates were separated by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the two-dimensional polyacrylamide gel electrophoresis patterns generated from the apoptotic cells did not differ dramatically from that of control cells. Thus, apoptotic Jurkat cells are able to synthesize new proteins and do not exhibit extensive proteolysis. Subsequent quantitative analysis revealed that only five proteins exhibited decreases in turnover that were common to the two treatments. No increases in protein turnover were able to be confirmed across the replicate experiments. One of the proteins that showed decreased labeling by both apoptotic inductions was an abundant nuclear protein with a pI of 5.1 and M(r) 40,000. This protein was identified as numatrin/B23/nucleophosmin (NPM) based on internal amino acid sequence, and this identity was confirmed by immunoblotting and mass spectrometry. NPM is implicated in a range of diverse cellular functions, but its role in apoptosis is unclear.
Patterson
S D
SD
Cold Spring Harbor Laboratory, New York 11724-2208, USA.
Grossman
J S
JS
D'Andrea
P
P
Latter
G I
GI
D017209:Apoptosis; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008148:Lovastatin / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D009687:Nuclear Proteins / Q000502:physiology*; D000090243:Nucleophosmin; D013601:T-Lymphocytes / Q000378:metabolism*
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D004533:Egtazic Acid; D008148:Lovastatin; D008715:Methionine; D003545:Cysteine