PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1.
BACKGROUND
The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). We have identified phosphatidic acid (PA) as a mediator of mitogenic activation of mTOR signaling. In this study, we set out to test the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1.


RESULTS
Overexpression of wild-type PLD1 increased S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerted a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi led to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also resulted in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action was demonstrated to be through the mTOR pathway. When Cdc42 was mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum was hindered. However, when exogenous PA was used as a stimulus, the PLD1-inactive Cdc42 mutant behaved similarly to the wild-type protein.


CONCLUSIONS
Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.
Fang
Yimin
Y
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Avenue B107, Urbana, IL 61801, USA.
Park
In-Hyun
IH
Wu
Ai-Luen
AL
Du
Guangwei
G
Huang
Ping
P
Frohman
Michael A
MA
Walker
Stephanie J
SJ
Brown
H Alex
HA
Chen
Jie
J
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D015870:Gene Expression; D008954:Models, Biological; D010739:Phospholipase D / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D002352:Carrier Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D058570:TOR Serine-Threonine Kinases; D010739:Phospholipase D; C106801:phospholipase D1; D020764:cdc42 GTP-Binding Protein