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

In the rat liver epithelial cell lines GN4 and WB, angiotensin II (Ang II) activates the G(q) class of regulatory G-proteins, increasing int racellular calcium, protein kinase C activity, and protein tyrosine ph osphorylation. We compared the ability of Ang II and other compounds t hat 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 (p70(S6K)) and p90 ribosomal S6 kinase (p90(RSK)). In GN4 cells, incre asing intracellular calcium stimulated p70(S6K) activity in a rapamyci n- and wortmannin- sensitive manner, but did not affect p90(RSK) activ ity. In contrast, 12-O-tetradecanoylphorbol-13-acetate strongly activa ted p90(RSK) but only weakly stimulated p70(S6K). The ability of calci um to activate p70(S6K) 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 p70( S6K) by Ang II, suggesting that this signal was largely calcium-depend ent. In contrast, the Ang II-dependent activation of mitogen-activated protein kinase and p90(RSK) was not inhibited but was enhanced by BAP TA-AM. These results show that in GN4 cells, Ang II selectively activa tes p70(S6K) through effects on calcium, p90(RSK) through effects on p rotein kinase C. The activation of p70(S6K) by calcium stimuli or Ang II was independent of calmodulin but correlated well with the activati on of the recently identified, nonreceptor calcium-dependent tyrosine kinase (CADTK)/PYK-2. Both calcium- and Ang II-dependent activation of p70(S6K) were attenuated by the tyrosine kinase inhibitor genistein, and activation of p70(S6K) was higher in GN4 than WB cells, correlatin g with the increased ex expression and activation of CADTK/PYK-2 in GN 4 cells. In summary, these results demonstrate that intracellular calc ium selectively activates p70(S6K) in GN4 cells, consistent with incre ased CADTK/PYK-2 signaling in these cells.