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Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells

1993; Elsevier BV; Volume: 268; Issue: 14 Linguagem: Inglês

10.1016/s0021-9258(18)82164-0

1083-351X

Ikuho Kojima, Hideo Mogami, Hiroshi Shibata, Etsuro Ogata,

Growth Hormone and Insulin-like Growth Factors

We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive Balb/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 phosphorylation 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 peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess 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 the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent 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.