Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells : lack of Rap 1 activation in variant cells

• Published in: Oncogene Vol. 19; no. 35; pp. 4029 - 4034
• Main Authors: VON LINTIG, F. C, PILZ, R. B, BOSS, G. R
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 17.08.2000; Nature Publishing Group
• Subjects: Animals; Biological and medical sciences; Cancer; Cancer cells; Cell activation; Cell differentiation; Cell Differentiation - drug effects; Cell Line; Cell physiology; Cellular signal transduction; Control; Cricetinae; Cyclic AMP; Cyclic AMP - analogs & derivatives; Cyclic AMP - pharmacology; Cyclic GMP; Cyclic GMP - analogs & derivatives; Cyclic GMP - pharmacology; Drug Resistance; Enzyme Activation; Enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genetic aspects; Guanine; Guanine nucleotide exchange factor; Guanine Nucleotide Exchange Factors - metabolism; Guanosine Triphosphate - metabolism; Health aspects; HL-60 Cells - drug effects; HL-60 Cells - metabolism; Humans; Kidney; Kinases; Leukemia; Mesocricetus; Methylation; Molecular and cellular biology; Neoplasm Proteins - metabolism; Peptides; Physiological aspects; Post-translation; Protein kinase A; Protein kinase inhibitors; Proteins; Proteolysis; rap1 GTP-Binding Proteins - metabolism; Rap1 protein; Recombinant Fusion Proteins - metabolism; Responses to growth factors, tumor promotors, other factors; Second Messenger Systems; Signal transduction; Thionucleotides - pharmacology; United States; United States > US; California; Human; Enzyme; Transferases; Rodentia; Cyclic AMP; 3',5'-GMP; Activation; HL60 cell line; Kidney; Signal transduction; Vertebrata; Mammalia; Cell line; Protein kinase; Nucleotide; Hamster; Quantitative analysis
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1203741

We have previously isolated variant HL-60 cells that are resistant to cGMP-induced differentiation and showed that they are deficient in proteolytic cleavage and/or carboxyl methylation of Rap 1A (J. Biol. Chem. 269, 32155 - 32161, 1994 and Oncogene 17, 2211 - 2233, 1998). We have now developed an enzyme-based method for assessing Rap 1 activation which is quantitative and provides a measurement of the per cent of Rap molecules in the active GTP-bound state. Using this method, we show that cAMP and cGMP analogs activate Rap 1 in parental HL-60 cells but not in the variant cells and that H-89, a cAMP-dependent protein kinase inhibitor, has no effect on cAMP-induced Rap 1 activation in parental cells. Thus, cAMP activation of Rap 1 in HL-60 cells is likely through a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) and since cAMP does not activate Rap 1 in the variant cells, the data suggest that full post-translational processing of Rap 1 is necessary for cAMP-GEF activation of Rap 1. Activation of Rap 1 by cGMP analogs has not been previously found and suggests possible cross-talk between the NO/cGMP signal transduction pathway and Rap 1 signaling. Oncogene (2000) 19, 4029 - 4034.