Catalytic activity of nuclear PLC- beta sub(1) is required for its signalling function during C2C12 differentiation

Here we report that PLC- beta sub(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC- beta sub(1) mutational analysis revealed the importance of His super(331) and His super(378) for the catalysis. The expression of PLC- beta sub(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC- beta sub(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC- beta sub(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC- beta sub(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC- beta sub(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC- beta sub(1) and that c-Jun binding activity is significantly increased by insulin and PLC- beta sub(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC- beta sub(1). These results hint at the fact that PLC- beta sub(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.