A systematic approach to the complete study of a signaling molecule: ribosomal p90(rsk) as an example

Ribosomal p90(rsk) is a kinase of central importance in transducing mitogen ic signals from an activated receptor to the cell nucleus and for protein s ynthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunolo gical analyses (immunoblotting and immunoprecipitation); (ii) enzyme activi ty assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" ass ay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90(rsk) kinase activity "i n-gel", based on a renatured-protein method that allows for the direct quan titation of enzyme activity. Finally, we present an algorithm that can be r eadily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli, In our case, it w as found that activation of p90(rsk) was higher in proliferating leukemic c ells than in mature neutrophils, indicating that a suppression of key signa l transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90(r sk) could be easily extrapolated to the study of any signal transduction mo lecule, provided it has a phosphotransferase activity. (C) 2001 Elsevier Sc ience B.V. All rights reserved.