Activation of ERK by Ca2+ store depletion in rat liver epithelial cells

In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two inde pendent methods resulted in activation of extracellular signal-regulated pr otein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargi n increased the activity of ERK, even when rise in cytosolic Ca2+ was block ed with the Ca2+ chelator BAPTA-AM. For the second method, addition of extr acellular EGTA at a concentration shown to deplete intracellular Ca2+ pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kina se inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasin g agent thapsigargin increased Fyn activity, which was unaffected by BAPTA- AM pretreatment, suggesting that Fyn activity was unaffected by increased c ytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphat ases. ANG II-induced activations of Fyn, Raf-l, and ERK were augmented in c ells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-sp ecificity phosphatase mitogen-activated protein kinase phosphatase-l was bl ocked by BAPTA-AM pretreatment. Together these results indicate that ERK ac tivity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+ stored in intracellu lar pools plays an important role in this regulation.