AN INTRACELLULAR CALCIUM SIGNAL ACTIVATES P70 BUT NOT P90 RIBOSOMAL S6 KINASE IN LIVER EPITHELIAL CELLS

Abstract In the rat liver epithelial cell lines GN4 and WB, angiotensin II (Ang II) activates the Gq class of regulatory G-proteins, increasing intracellular calcium, protein kinase C activity, and protein tyrosine phosphorylation. We compared the ability of Ang II and other compounds that increase intracellular calcium (i.e. the calcium ionophore A23187 and thapsigargin) or protein kinase C activity (the phorbol ester 12-O-tetradecanoylphorbol-13-acetate) to activate p70 ribosomal S6 kinase (p70S6K) and p90 ribosomal S6 kinase (p90RSK). In GN4 cells, increasing intracellular calcium stimulated p70S6K activity in a rapamycin- and wortmannin- sensitive manner, but did not affect p90RSK activity. In contrast, 12-O-tetradecanoylphorbol-13-acetate strongly activated p90RSK but only weakly stimulated p70S6K. The ability of calcium to activate p70S6K was confirmed by blocking the A23187-dependent activation through chelation of extracellular calcium with EGTA; the effect of thapsigargin was inhibited by the cell permeant chelator bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Similarly, BAPTA-AM prevented the activation of p70S6K by Ang II, suggesting that this signal was largely calcium-dependent. In contrast, the Ang II-dependent activation of mitogen-activated protein kinase and p90RSK was not inhibited but was enhanced by BAPTA-AM. These results show that in GN4 cells, Ang II selectively activates p70S6K through effects on calcium, p90RSK through effects on protein kinase C. The activation of p70S6K by calcium stimuli or Ang II was independent of calmodulin but correlated well with the activation of the recently identified, nonreceptor calcium-dependent tyrosine kinase (CADTK)/PYK-2. Both calcium- and Ang II-dependent activation of p70S6K were attenuated by the tyrosine kinase inhibitor genistein, and activation of p70S6K was higher in GN4 than WB cells, correlating with the increased expression and activation of CADTK/PYK-2 in GN4 cells. In summary, these results demonstrate that intracellular calcium selectively activates p70S6K in GN4 cells, consistent with increased CADTK/PYK-2 signaling in these cells.