Signal Transduction in Fibroblasts Stably Transformed by [Val12]Ras

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Abstract
Ras‐transformed cells often show high levels of expression of activating protein‐1 and Ets and of genes regulated by these transcription factors. In analogy with the effects of transient stimulation of Ras, it is assumed that the increase in transcription‐factor trans activation in stably transformed cells is due to Ras‐induced constitutive activation of mitogen‐activated protein kinases. However, this has not been extensively studied. Using specific substrate peptides, we have examined here the activities of two types of mitogen‐activated protein kinase, extracellular‐signal‐regulated kinase (ERK) and Jun N‐terminal kinase (JNK), in [Val12]Ras‐transformed rat embryo fibroblast cell lines. These activities were elevated 2–3‐fold in Ras‐transformed cells compared with non‐transformed cells with a similar growth rate. Increased ERK activity was not necessarily accompanied by a similar increase in JNK activity. In transformed cells, ERK and JNK activities could be stimulated fourfold and ninefold by phorbol ester and ultraviolet‐light treatment, respectively, indicating that only a fraction of these enzymes were constitutively activated in these cells. It has been suggested that inactive JNK downregulates c‐Jun transcriptional activity by binding to the c‐Jun δ‐domain. No decrease in δ‐inhibitor activity could be demonstrated in Ras‐transformed cells compared with control cells, consistent with the presence of mainly inactive JNK in transformed cells. Treatment of transformed cells with benzodiazepine 5B, an inhibitor of Ras farnesylation, decreased ERK and JNK activities, and concomitantly caused morphological reversion, reduced growth rate, and normalization of transformation‐related gene expression. We conclude that in stably Ras‐transformed cells the moderately increased ERK/JNK activities are not coregulated, and that ERK rather than JNK activity correlated with transformation‐related gene expression.