Abstract
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.
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Acknowledgements
We would like to thank Dr J Bos for generously providing the GST-RBD expression vector. This work was supported in part by US Army Grant #DAMD17-97-1-7031 (to GR Boss) and by NIH Grant R01GM055586 (to RB Pilz); FvL was supported by the Mildred Scheel-Stiftung für Krebsforschung and by National Institutes of Health Grant #R21 CA81115.
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von Lintig, F., Pilz, R. & Boss, G. Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells. Oncogene 19, 4029–4034 (2000). https://doi.org/10.1038/sj.onc.1203741
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DOI: https://doi.org/10.1038/sj.onc.1203741
