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HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells
Published on: July 20, 2016
Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1
F C von Lintig1, R B Pilz, G R Boss
1Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California, CA 92093-0652, USA.
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. Oncogene (2000) 19, 4029 - 4034.
Insights
Variant HL-60 cells resistant to differentiation lack Rap 1 activation. This study introduces a method to measure Rap 1 activation, revealing that post-translational processing is crucial for cAMP-GEF signaling and suggesting cross-talk with cGMP pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Signal Transduction
Background:
- Previously identified HL-60 cell variants resistant to cyclic guanosine monophosphate (cGMP)-induced differentiation exhibit defects in Rap 1A post-translational modification.
- Rap 1A is a small GTPase involved in various cellular processes, including differentiation and signaling pathways.
Purpose of the Study:
- To develop a quantitative method for assessing Rap 1 activation.
- To investigate the role of Rap 1 post-translational processing in cyclic adenosine monophosphate (cAMP) and cGMP signaling.
- To explore the mechanism of cAMP-induced Rap 1 activation in HL-60 cells.
Main Methods:
- Development of an enzyme-based assay to quantify the GTP-bound, active state of Rap 1.
- Treatment of parental and variant HL-60 cells with cAMP and cGMP analogs.
- Assessment of Rap 1 activation in response to signaling molecules and a protein kinase inhibitor (H-89).
Main Results:
- Parental HL-60 cells showed Rap 1 activation by cAMP and cGMP analogs, while variant cells did not.
- cAMP-induced Rap 1 activation in parental cells was independent of cAMP-dependent protein kinase.
- The findings suggest that full post-translational processing of Rap 1 is essential for activation by cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs).
Conclusions:
- cAMP-mediated Rap 1 activation in HL-60 cells likely involves a cAMP-GEF and requires complete Rap 1 post-translational modification.
- The observed activation of Rap 1 by cGMP analogs suggests a potential cross-talk between the nitric oxide/cGMP pathway and Rap 1 signaling.
- Defects in Rap 1 processing impair its activation, highlighting the importance of post-translational modifications in signal transduction pathways.
