An investigation into the cellular functions of ERK1/ERK2 and PTPa using antisense oligodeoxynucleotides

Antisense oligodeoxynucleotides (ODNs) are potent inhibitors of gene expression. The present work has involved targeting several cellular signalling proteins, namely the extracellular signal-regulated protein kinases-1 and -2 (ERK1/ERK2) and protein tyrosine phosphatase-α (PTPα), with specific phosphorothioate ODNs in order to examine the potential physiological roles of these proteins.ERK1/ERK2 have previously been implicated as candidates for the growth factor- and insulin-stimulated phosphorylation of PHAS-I. A specific antisense strategy against ERK1/ERK2 was utilised to determine whether ERK1/ERK2 mediate in foetal serum (FBS)-stimulated PHAS-I phosphorylation in intact 3T3-L1 adipocytes. Depleting >90% of cellular ERK1/ERK2 had no effect on FBS-stimulated PHAS-I phosphorylation. However, treatment of cells with a specific p70S6k pathway inhibitor, rapamycin, markedly attenuated PHAS-I phosphorylation in response to FBS. These results suggest that FBS-stimulated PHAS-I phosphorylation occurs through an ERK1/ERK2-independent and rapamycin-sensitive pathway, in 3T3-L1 adipocytes. Similarly, insulin-stimulated PHAS-I phosphorylation was shown to be rapamycin-sensitive.Antisense strategies were developed capable of specifically depleting PTPα from intact 3T3-L1 fibroblasts and adipocytes. Steady state expression of PTPα protein was reproducibly inhibited by ≈85% in both cell types, as determined by Western blot. PTPα depletion caused a significant reduction in the level of pp60c-src activity in 3T3-L1 adipocytes. Moreover, treatment of 3T3-L1 fibroblasts with the antisense probe prevented their differentiation to adipocytes. In contrast, depleting PTPα from 3T3-L1 adipocytes had no effect on the tyrosine phosphorylation state of the insulin receptor tyrosine kinase or the major insulin receptor substrate proteins, termed pp185. Furthermore, several post-receptor responses (namely insulin- stimulated ERK1/ERK2 activation and DNA synthesis) were not affected by the decrease in PTPα expression.This work demonstrates that antisense strategies are invaluable tools for the study of specific components of cellular signalling pathways, providing new insights into the mechanisms by which cellular signals are regulated.