The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis.
Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD-PX are vital for this GAP function. Moreover, wild-type PLD-PX, but not mutated PLD-PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity - a novel function in addition to its role as a binding motif for proteins or lipids.
Lee
Chang S
CS
Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, South Korea.
Kim
Il S
IS
Park
Jong B
JB
Lee
Mi N
MN
Lee
Hye Y
HY
Suh
Pann-Ghill
PG
Ryu
Sung H
SH
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D020690:GTPase-Activating Proteins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010739:Phospholipase D; D034281:Dynamins