A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example.
Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.
Sampt
E R
ER
Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Fernandez
G A
GA
Lehman
J A
JA
Corey
S J
SJ
Huang
C K
CK
Gómez-Cambronero
J
J
D000465:Algorithms; D001671:Biochemistry / Q000379:methods*; D002454:Cell Differentiation; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology; D011233:Precipitin Tests; D011489:Protein Denaturation; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry*; D013379:Substrate Specificity; D013997:Time Factors
D019893:Ribosomal Protein S6 Kinases