Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells.
Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.
Powner
Dale J
DJ
Institute for Cancer Studies, Birmingham University, Birmingham, B15 2TA, United Kingdom.
Hodgkin
Matthew N
MN
Wakelam
Michael J O
MJ
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010739:Phospholipase D / Q000378:metabolism*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat; D020830:rac1 GTP-Binding Protein