PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1

Curr Biol. 2003 Dec 2;13(23):2037-44. doi: 10.1016/j.cub.2003.11.021.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • COS Cells
  • Carrier Proteins / metabolism*
  • Gene Expression
  • Models, Biological
  • Phospholipase D / metabolism*
  • Phosphoproteins / metabolism*
  • Precipitin Tests
  • Protein Kinases / metabolism*
  • Protein Kinases / physiology
  • Ribosomal Protein S6 Kinases, 90-kDa / metabolism*
  • Signal Transduction / physiology*
  • TOR Serine-Threonine Kinases
  • cdc42 GTP-Binding Protein / metabolism*

Substances

  • Carrier Proteins
  • Phosphoproteins
  • Protein Kinases
  • TOR Serine-Threonine Kinases
  • Ribosomal Protein S6 Kinases, 90-kDa
  • Phospholipase D
  • phospholipase D1
  • cdc42 GTP-Binding Protein