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* Cell Biology and Physiology, Wright State University School of Medicine, Dayton, Ohio, USA; and
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA
1Correspondence: Department Neuroscience, Cell Biology and Physiology, Wright State University, School of Medicine, 3640 Colonel Glenn Hwy., Dayton, Ohio 45435, USA. E-mail: julian.cambronero{at}wright.edu
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.—Lehman, N., Ledford, B., Di Fulvio, M., Frondorf, K., McPhail, L. C., Gomez-Cambronero, J. Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR.
Key Words: signal transduction • network phospholipases and kinases • small interfering RNA • protein/lipid binding • immunofluorescence colocalization
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