GTP-dependent interaction between phospholipase D and dynamin modulates fibronectin-induced cell spreading.
Phospholipase D (PLD) is one of the key enzymes to mediate a variety of cellular phenomena including endocytosis, actin rearrangement, proliferation, differentiation, and migration. Dynamin as a PLD-interacting partner is a large GTP binding protein that has been considered a mechanochemical enzyme involved in endocytosis by hydrolyzing GTP. Although both PLD and dynamin have been implicated in the regulation of actin cytoskeleton, it is not known how they have a link to regulate fibronectin (FN)-induced cell spreading. Furthermore, it is unknown whether dynamin can work as a GTP-dependent regulator through its interaction with other proteins. Here, we demonstrate that PLD can be regulated by dynamin in a GTP-dependent manner and that this is critical for FN-mediated cell spreading. First, we verified that GTP-loaded dynamin can mediate the cell spreading by FN by using dynamin's GTP binding deficient mutant (K44A). Also, we confirmed that blocking the PLD activity inhibited FN-induced cell spreading, not cell adhesion. Moreover, PLD interacted with dynamin in a GTP-dependent manner in FN signaling, and this interaction was crucial for FN-induced PLD activation and cell spreading. Also, we found that PLD mutant (R128K) that didn't have GAP activity increased the GTP-dependent interaction between PLD and dynamin; it also increased PLD activity and cell spreading. These findings suggest that the observed increase in PLD activity was through boosting the binding of PLD with dynamin and it facilitated FN-induced cell spreading. These results imply that GTP-loaded dynamin, like a small GTPase could mediate a "switch on" signaling via interaction with PLD that has a role as an effector.
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea.
Chang Sup
CS
Lee
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea.
Jong Min
JM
Kim
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea.
Jaewang
J
Ghim
School of Life Science, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, South Korea.
Pann-Ghill
PG
Suh
Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea. Electronic address: sungho@postech.ac.kr.
Sung Ho
SH
Ryu
D002448:Cell Adhesion; D048430:Cell Shape; D034281:Dynamins / Q000378:metabolism*; D004789:Enzyme Activation; D005353:Fibronectins / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D010739:Phospholipase D / Q000378:metabolism*
D005353:Fibronectins; D006160:Guanosine Triphosphate; D010739:Phospholipase D; D034281:Dynamins
Dynamin; GTPase; GTPase activating protein; Interaction; Phospholipase D; Spreading