Protein kinase C α trigger Ras and Raf-independent MEK/ERK activation for TPA-induced growth inhibition of human hepatoma cell HepG2

Abstract

The tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) is a well-known activator of both protein kinase C (PKC) and mitogen activated protein kinase (MAPK) signal cascade triggering a lot of effects in many non-tumor and tumor cells. We have reported activation of PKCα isozyme was specifically required for TPA-induced ERK (MAPK) signaling that mediated gene expressions of the CDK inhibitors p15 INK4b and p16 INK4a leading to growth inhibition of hepatoma cell HepG2. We further investigated the upstream signal molecule linking PKCα to ERK. In the Ras activation assay, HepG2 cell exhibited substantial amount of Ras activity. Treatment of the cell with 50 nM TPA for 10 min slightly inhibited Ras activity by about 10–20%. Pretreatment of the cell with 10 μM manumycin A, which abolish basal Ras activity, did not prevent TPA-triggered ERK phosphorylation. Immunoprecipitation coupled with kinase assay demonstrated that MEK-1 activity was strongly induced by treatment of TPA for 5–30 min in HepG2. In contrast, c-Raf activity was not significantly induced by TPA within 5–15 min. Consistently, Western blot of Phospho(ser-218/222)-MEK demonstrated that phosphorylation of MEK-1 was greatly induced by 50 nM TPA, which can be prevented by the PKC inhibitor Bisindolylmaleimides II. Moreover, pretreatment of the MEK1/2 inhibitor, but not c-Raf inhibitor prevented the TPA-induced ERK phosphorylation, gene expression of p15 INK4b and p16 INK4a and growth inhibition of HepG2. In addition, transient expression of a dominant negative Raf mutant in HepG2 did not prevent these effects of TPA. Constitutive expression of an active PKCα mutant in HepG2 enhanced phosphorylation of both MEK and ERK accompanied with induction of gene expression of p16 INK4a and growth inhibition of HepG2. In contrast, Ras and Raf activity were not increased by expression of active PKCα. Taken together, we conclude that PKCα may activate MEK, independently of Raf and Ras, to trigger sustained ERK (MAPK) signaling and cell cycle arrest of HepG2 induced by TPA.