A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein

Abstract

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assess- ment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the tran- scription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflam- matory prostaglandins on CREB phosphorylation in macro- phages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.