Phospholipase D (PLD) catalyzes the production of phosphatidic acid (PA) from phosphatidylcholine. Proteins with specific motifs recognize PA and may be recruited to the site of its production. In addition, PA may be further hydrolyzed to produce diacylglycerol (DAG), another lipid to which proteins may interact. Two reports find that PLD and PA are involved in the activation of Ras at the plasma membrane (see Hancock for discussion). Although SOS (son of sevenless), the protein that activates Ras in response to receptor tyrosine kinase activation, binds to the receptor-associated Grb2, this interaction is not required for recruitment of SOS to the membrane. Instead, Zhao et al. found that the pleckstrin homology (PH) domain of SOS bound specifically to PA with high affinity in vitro. Disruption of this interaction and the interaction with Grb2 in the same SOS construct (SosΔCHR/EE) eliminated the recruitment of the transfected SOS to the membrane in response to the addition of PA or serum (to stimulate receptor tyrosine kinases). Furthermore, in response to epidermal growth factor (EGF), Ras activation occurred in cells expressing the Grb2-binding mutant SosΔC but did not occur if both the Grb2 and PA interactions were abolished. When PLD2, which interacts with EGF receptor-Grb2 complexes, was knocked down by RNA interference techniques, EGF-stimulated activation of Ras was blocked. Thus, Zhao et al. suggest that the interaction of SOS with PLD-produced PA is the critical step in recruiting SOS to the membrane to activate Ras and that Grb2 serves to recruit PLD2 to the activated receptors.
Activation of the T cell receptor (TCR) alone or with its co-receptor activates Ras only at the Golgi, and this Ras activation involves the DAG- and calcium-regulated guanine nucleotide releasing protein RasGRP1. Mor et al. show that coactivation of the TCR and the integrin LFA-1 (lymphocyte function-associated antigen-1, also known as αLβ2) results in activation of Ras at the plasma membrane and Golgi and implicate PLD2 in the plasma membrane response. Live-cell imaging was performed with Jurkat T cells expressing green fluorescent protein (GFP)-tagged probes for activated Ras or DAG along with tagged versions of the three isoforms of Ras (N- and H-Ras, which cycle between the Golgi and plasma membrane, and K-Ras, which is recruited only to the plasma membrane). Only when both the TCR and LFA-1 were engaged was Ras activated at the plasma membrane. Endogenous K-Ras was only activated when both the TCR and LFA-1 were engaged. Cells deficient in phospholipase Cγ1 blocked Ras activation at the Golgi but did not inhibit Ras activation at the plasma membrane in response to dual TCR and LFA-1 engagement. In contrast, inhibition of PLD with low concentrations of n-butanol blocked recruitment of RasGRP1 to the plasma membrane and Ras activation at the plasma membrane; higher concentrations of n-butanol blocked Ras activation at the Golgi as well. Inhibition of phosphatidic acid phosphatase (PAP), which is the enzyme responsible for producing DAG from PA, also blocked Ras activation at the plasma membrane. A DAG fluorescent reporter showed that DAG concentrations increased at the plasma membrane in response to TCR and LFA-1 engagement. These results suggest that PLD may shape the pattern of the T cell response.
C. Zhao, G. Du, K. Skowronek, M. A. Frohman, D. Bar-Sagi, Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. Nat. Cell Biol. 9, 706-712 (2007). [PubMed]
A. Mor, G. Campi, G. Du, Y. Zheng, D. A. Foster, M. L. Dustin, M. R. Philips, The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2. Nat. Cell Biol. 9, 713 -719 (2007). [PubMed]
J. F. Hancock, PA promoted to manager. Nat. Cell Biol. 9, 615 -617 (2007). [PubMed]