Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells.
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.
von Lintig
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FC
Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California, CA 92093-0652, USA.
Pilz
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Boss
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D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D004351:Drug Resistance; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007668:Kidney; D008647:Mesocricetus; D009363:Neoplasm Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D013873:Thionucleotides / Q000494:pharmacology; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins; D006152:Cyclic GMP