INSULIN AND TUMOR-PROMOTING AGENT REGULATE AN INHIBITOR OF THE 44-KDAMITOGEN-ACTIVATED PROTEIN-KINASE EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE-1 IN FIBROBLASTS

We have examined the negative regulation of the 44-kDa mitogen-activat ed protein kinase (MAP kinase), also known as extracellular signal-reg ulated protein kinase 1 (ERK1), in NIH3T3 cells transfected with an ex pression plasmid encoding the human insulin receptor (NHIR cells). In these cells ERK1 activation is induced by two distinct stimuli, insuli n and tumor-promoting agent (TPA). While insulin was found to be more potent than TPA for ERK1 activation, both stimuli produced the same tr ansient activation pattern with a rapid peak (reached within 5 min) fo llowed by a fast decrease within 20 min. By performing reconstitution experiments with immunoprecipitated ERK1 and lysates from NHIR cells, we showed that extracts from untreated cells exhibit an ERK1 inhibitor y activity. Interestingly, this inhibitor was found to be regulated by insulin and TPA with a profile that is the mirror image of ERK1 activ ity. This repressing activity was sensitive to tyrosine phosphatase in hibitors, such as sodium orthovanadate and zinc acetate, but it was no t affected by serine/threonine phosphatase inhibitors, such as sodium fluoride and okadaic acid. Moreover, it was possible to observe in ext racts of NHIR cells an activity dephosphorylating ERK1. The time cours e of this phosphatase activity was comparable to that of the ERK1 inhi bition, suggesting that the repressing activity could reflect a dephos phorylating action. Interestingly, phosphatase 2A treatment of extract s from 5-min TPA-treated cells (where the ERK1 inhibitor was weak) was able to induce an increase in the ERK1 repressing activity. This sugg ests that serine/threonine dephosphorylation of ERK1 inhibitor leads t o an increase in its activity. In summary, we have shown that NHIR cel ls contain a regulatable ERK1 inhibitor, which is likely to be due to tyrosine phosphatase(s). We would like to suggest that such activities are key components in the fine-tuning of the MAP kinase cascade.