RNAi and 2DE, a promising combination for analysis of phospho-signalling and substrate identification

The use of RNA interference (RNAi) has provided the study of cell signalling with a specific and relatively easily applicable method of silencing individual components of signalling pathways. RNAi mediated gene muting was combined with two-dimensional electrophoresis (2DE) and immuno-blotting analysis (IB) to study phospho-signalling downstream of the protein kinase, Akt. NIH3T3 mouse fibroblasts were transiently transfected with short interfering RNAs (siRNAs) to Akt with resulting suppression of Akt expression. Down-regulation of Akt reduced the cellular content of phosphorylated FKHR, enhanced GSK3β phosphorylation, and increased the sensitivity of the cells to apoptotic agents. Stable transfection of short hairpin RNAs inserted into a stable expression vector, pRETRO-SUPER (pRS), also proved to be a successful method of downregulating Akt1, and resulted in an altered phosphorylation pattern and a reduced rate of cell proliferation. Akt-regulated phosphorylation was identified by comparison of extracts from Akt RNAi transfected cells with extracts from cells transfected with pRS vector alone. While Akt muting suppressed some phospho-modifications, others were enhanced. The␣PDGF-induced tyrosine phosphorylation cascades were, surprisingly, found to be influenced by RNAi-mediated Akt muting. Tyrosine phosphorylation of some proteins were reduced in cells with down-regulated Akt1 activity, suggesting the existence of either a downstream tyrosine kinase positively regulated by Akt1, or a negatively regulated tyrosine phosphatase, positioned centrally in the cross-talk between the Akt1 regulated signalling pathways and the growth factor induced phospho-tyrosine pathways. Our results illustrate the analytical potential of combining RNAi-mediated regulator muting with 2DE-based analysis of phospho-signalling, and suggest that such a combination could become a highly efficient tool for the identification and characterisation of new kinase substrates.