Phospholipase C β3 is a key component in the Gβγ/PKCη/PKD-mediated regulation of trans-Golgi network to plasma membrane transport

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric G beta gamma (GTP-binding protein beta gamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKC eta (protein kinase C eta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PK07, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by G beta gamma, utilizing PtdIns(4,5)P-2 as a substrate, to produce the second messengers DAG and InSP3. In the present study we show that beta gamma-dependent Golgi fraginentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-f{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}-1 hexyl)-1-Hpyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLC 3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLC 3, which is necessary to activate PKC eta and PKD in that Golgi compartment, via DAG production.