ROLE OF CALCIUM ENTRY AND PROTEIN-KINASE-C IN THE PROGRESSION ACTIVITY OF INSULIN-LIKE GROWTH FACTOR-I IN BALB C 3T3 CELLS/

We previously reported that insulin-like growth factor-I (IGF-I) stimu lated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E ., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and produ ction of 1,2-diacylglycerol in IGF-responsive ''primed competent'' Bal b/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphory lation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF- I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR pepti de phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibi tor of PKC. The phosphorylation remained elevated for up to 6 h. To as sess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of t he G1 phase, but did not affect cell cycle progression when added at t he second quarter or later. In contrast, reduction of extracellular ca lcium completely blocked cell cycle progression when done in the first , second, and third quarter but had no effect in the last quarter. The se results indicate that IGF-I persistently activates PKC in primed co mpetent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.