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Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon
B Francis1, J Overmeyer, W John
1Lucille P. Markey Cancer Center, University of Kentucky, Lexington.
Abstract:
The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.
Insights
Nucleoside diphosphate kinase (NDPK) activity is crucial for cellular events requiring GTP. A new method shows a specific NDPK form is tenfold higher in colon tumors, indicating elevated NDPK activity in malignancy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- G-regulatory proteins, tubulin, and ras oncogene protein require GTP for cellular function.
- Nucleoside diphosphate kinase (NDPK) is critical for producing GTP from ATP, regulating GTP-dependent cellular events.
- The activity level of NDPK is a key factor in cellular regulation.
Purpose of the Study:
- To develop a method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK ~P).
- To investigate the role of NDPK ~P levels in colon tumor tissues compared to normal tissues.
- To characterize the autophosphorylation and dephosphorylation mechanisms of NDPK.
Main Methods:
- Developed a method using EDTA to trap NDPK in its high-energy phosphorylated form (NDPK ~P).
- Utilized [gamma-32P]ATP, [gamma-32P]GTP, and 8-azidopurine photoprobes for autophosphorylation studies.
- Employed Western blot and photoaffinity labeling to distinguish NDPK ~P subunits from other GTP-binding proteins.
Main Results:
- NDPK ~P levels were found to be tenfold higher in malignant colon tumor tissue than in normal colon tissue.
- Autophosphorylation of NDPK subunits (21,000 and 24,000 Mr) occurred rapidly at 0°C and was intramolecular.
- Assays confirmed correspondingly elevated NDPK activity in malignant homogenates, suggesting NDPK ~P is a quantitative measure of the specific isozyme form.
Conclusions:
- The study presents a novel method for measuring a specific NDPK isozyme form.
- Elevated levels of NDPK ~P and overall NDPK activity in colon tumors suggest a significant role in cancer.
- NDPK ~P levels may serve as a quantitative biomarker for malignant colon tissue.