Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/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 Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) 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 PKCeta, 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 Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-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 PLCbeta3 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 PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.
Díaz Añel
Alberto M
AM
Center for Molecular Genetics, University of California at San Diego, San Diego, CA, USA. adiazanel@immf.uncor.edu
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004963:Estrenes / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits / Q000302:isolation & purification / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D054799:Phospholipase C beta; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011760:Pyrrolidinones / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013449:Sulfonamides; D010738:Type C Phospholipases / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism*; D000096926:Benzenesulfonamides
D004075:Diglycerides; D004963:Estrenes; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D054799:Phospholipase C beta; D011493:Protein Kinase C; D011760:Pyrrolidinones; D034741:RNA, Small Interfering; D013449:Sulfonamides; D010738:Type C Phospholipases; D014759:Viral Envelope Proteins; C473783:2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide; D000096926:Benzenesulfonamides; C028391:G protein, vesicular stomatitis virus; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C081975:protein kinase C eta; C089212:protein kinase D; C518039:PLCB3 protein, human; C518041:Plcb3 protein, rat