Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

Abstract

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamycin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry of 94:1 lipid/protein. Polyphosphoinositides PI4-P and PI4,5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS-7 cells with a wild-type myc-(pcDNA)-PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6K's natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS-7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR-independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.

Phosphatidic Acid Is a Leukocyte Chemoattractant That Acts through S6 Kinase Signaling

“…Exposure of cells to PA resulted in the accumulation of PA inside the cell clustered into discrete patches/vesicles where it can lead to the activation of S6K. Lehman et al (6) have shown immunofluorescent staining of S6K shifting away from perinuclear regions and colocalizing with PLD2 in the cytosol. It is also possible that PA could be on early endosomes during triggered endocytosis and subsequently move into recycling endosomes.…”

mTOR signaling: PLD takes center stage

“…For instance, overexpression of recombinant PLD2 has been reported to activate mTORC1 in MCF-7 breast cancer cells 25 as well as COS-7 cells. 28,36 It is noteworthy that in the Lehman et al, report recombinant PLD2 is shown to co-localize with S6K1 in the perinuclear region. 28 This contradicts the predominant plasma membrane localization of PLD2 observed by others, 69,71 and raises a concern for potential artifact of protein overexpression.…”

“…23,27 However, the mechanism by which PA activates mTOR remains unknown. Curiously, Lehman et al, 28 reported direct binding and activation of S6K1 by PA in vitro, suggesting a route of PA regulation that is independent of mTOR.…”

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

“…The two main substrates of mTORC1 are the translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), whereas mTORC2 phosphorylates PKB on serine 473. In addition, regulation of the mTORC1 pathway through the binding of the lipid messenger phosphatidic acid (PA) to mTOR [12,13] and S6K [14] has been described. PA is produced through the activity of phospholipase D (PLD) isozymes, among which PLD2 has been shown to be concentration-dependently regulated by oleate [15].…”