The co-mitogenic combination of transforming growth factor β₁ and bombesin protects protein kinase C-δ from late-phase down-regulation, despite synergy in diacylglycerol accumulation

Bombesin induces the down-regulation of protein kinase C-Δ (PKC-Δ) and PKC-ϵ in Swiss 3T3 cells. Simultaneous addition of transforming growth factor β₁ (TGFβ₁) selectively blocks PKC-Δ down-regulation at mid-S-phase, whereas PKC-ϵ levels continue to decline. Northern blot analysis shows that PKC-ϵ levels could be controlled in part at the level of transcription; PKC-Δ mRNA levels remained constant at these later times. Bombesin induces a sustained elevation of some species of diacylglycerol (DAG), consistent with the observed loss of PKC-Δ and PKC-ϵ. Interestingly, the combination of bombesin and TGF-β₁ produces an even greater DAG response. Flow cytometric analysis demonstrates that bombesin induces only 15% of the cells to enter the cell cycle, in contrast to the combination of TGFβ₁ plus bombesin which induces 75–80% of the cells to progress through the cycle. The protection of PKC-Δ from down-regulation under conditions of sustained DAG elevation correlates with the mitogenic response and implies that the down-regulation process itself is regulated. Consistent with this, it is demonstrated that bombesin plus TGFβ₁ protects PKC-Δ from phorbol ester-induced down-regulation.