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.