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.
a b s t r a c tJanus kinase 3 (JAK3) is a non-receptor tyrosine kinase vital to the regulation of T-cells. We report that JAK3 is a mediator of interleukin-8 (IL-8) stimulation of a different class of hematopoietic relevant cells: human neutrophils. IL-8 induced a time-and concentration-dependent activation of JAK3 activity in neutrophils and differentiated HL-60 leukemic cells. JAK3 was more robustly activated by IL-8 than other kinases: p70S6K, mTOR, MAPK or PKC. JAK3 silencing severely inhibited IL-8-mediated chemotaxis. Thus, IL-8 stimulates chemotaxis through a mechanism mediated by JAK3. Further, JAK3 activity and chemotaxis were inhibited by the flavonoid apigenin (4 0 ,5,7-trihydroxyflavone) at $5 nM IC 50 . These new findings lay the basis for understanding the molecular mechanism of cell migration as it relates to neutrophil-mediated chronic inflammatory processes.
We report here that the enzymatic activity of phospholipase D2 (PLD2) is regulated by phosphorylationdephosphorylation. 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 Phospholipase D2 (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger phosphatidic acid (PA) and choline. There are two mammalian isoforms: PLD1 (20) and PLD2 (12, 33). They share a highly conserved "HKD" domain necessary for catalysis (39). PLD has been implicated in a variety of physiological cellular functions, such as intracellular protein trafficking, cytoskeletal dynamics, membrane remodeling, and cell proliferation in mammalian cells and meiotic division and sporulation in yeast (6,15,16,32,42). PLD regulation in cells falls into two major signaling categories. One is by the small GTPase proteins Arf and Rho (3,5,11,43), and the other is by growth factors/ mitogens such as epidermal growth factor (EGF), plateletderived growth factor, insulin, and serum that implicate tyrosine kinases (2,12,21,22,34). Several gaps in knowledge of the second arm remain in spite of its importance in cell signaling. The isoform PLD1 can be phosphorylated on tyrosine, but this does not lead to changes in activity (35, 36).The isoform PLD2, which is expressed as a constitutively active enzyme in many cell types (47), can be detected as a phosphotyrosine protein both in vivo (25) and in vitro (17,25,41). PLD2 overexpression induces the transformation of cells (27,34,37,48), and the activation of this enzyme enhances cellular processes favorable for the metastasis of EL4 lymphoma cells (30). PLD also activates STAT3 by interacting with the thyroid oncogenic kinase RET/PTC (29). PLD2 can form a complex with the EGF receptor (EGFR) (12, 46) or with Pyk2 and Src kinases (4). Hydrogen peroxide and EGF induce PLD phosphorylation and PKC-␣ activation (38), while activation of PLD by 8-Br-cAMP is accomplished through Src, Ras, and ERK (49).Although all of these studies agree with respect to the presence of tyrosine kinases in PLD signaling, different conclusions were drawn regarding lipase activity. For example, whereas coexpression of PLD2 and Fyn or Fgr kinases lead to an enhancement of PLD activation and degranulation of mast cells (10), Ho et al. (23) , and Y 470 are important for PLD2 function (10) but, nevertheless, the overexpression of the phosphorylation-deficient mutant, PLD2-Y11F, increases PLD2 activity in resting or EGFactivated cells (12).Closely related to the enhancing effect of phosphorylationdeficient mutants, a new line of rese...
The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the lipase's functionality in vivo. In this study, we demonstrate that stable shRNA-based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2 SiL ) into the Grb2-knockdown cells (COS7 shGrb2 ), resulted in the nearly full rescue of PLD2 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7 shGrb2 cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespectively of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments of Grb2 and PLD2-Y169/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGF. In summary, these results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo.
We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by ϳ60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid , known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.
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