A Comprehensive Model That Explains the Regulation of Phospholipase D2 Activity by Phosphorylation-Dephosphorylation

We report here that the enzymatic activity of phospholipase D2 (PLD2) is regulated by phosphorylation-dephosphorylation. Phosphatase treatment of PLD2-overexpressing cells showed a biphasic nature of changes in activity that indicated the existence of "activator" and "inhibitory" sites. We identified three kinases capable of phosphorylating PLD2 in vitro-epidermal growth factor receptor (EGFR), JAK3, and Src (with JAK3 reported for the first time in this study)-that phosphorylate an inhibitory, an activator, and an ambivalent (one that can yield either effect) site, respectively. Mass spectrometry analyses indicated the target of each of these kinases as Y-296 for EGFR, Y-415 for JAK3, and Y-511 for Src. The extent to which each site is activated or inhibited depends on the cell type considered. In COS-7, cells that show the highest level of PLD2 activity, the Y-415 is a prominent site, and JAK3 compensates the negative modulation by EGFR on Y-296. In MCF-7, cells that show the lowest level of PLD2 activity, the converse is the case, with Y-296 unable to compensate the positive modulation by Y-415. MTLn3, with medium to low levels of lipase activity, show an intermediate pattern of regulation but closer to MCF-7 than to COS-7 cells. The negative effect of EGFR on the two cancer cell lines MTLn3 and MCF-7 is further proven by RNA silencing experiments that yield COS-7 showing lower PLD2 activity, and MTLn3 and MCF-7 cells showing an elevated activity. MCF-7 is a cancer cell line derived from a low-aggressive/invasive form of breast cancer that has relatively low levels of PLD activity. We propose that PLD2 activity is low in the breast cancer cell line MCF-7 because it is kept downregulated by tyrosyl phosphorylation of Y-296 by EGFR kinase. Thus, phosphorylation of PLD2-Y-296 could be the signal for lowering the level of PLD2 activity in transformed cells with low invasive capabilities.