Identification of the Catalytic Site of Phospholipase D2 (PLD2) Newly Described Guanine Nucleotide Exchange Factor Activity

Journal of Cell Science

Henkels

Gómez‐Cambronero

2013

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SummaryPhospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ,3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at .15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF-lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

Section: Introductionmentioning

confidence: 98%

A GEF-to-phospholipase molecular switch caused by PA, RAC and jak tyrosine kinase, that explains leukocyte cell migration

Journal of Cell Science

Henkels

Gómez‐Cambronero

2013

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“…3 H]butanol as previously described (24)(25)(26). Approximately 50 g of cell sonicates was added to microcentrifuge Eppendorf tubes containing the following assay mix (120-l final volume): 3.5 mM PC8 phospholipid, 1 mM PIP2, 75 mM HEPES, pH 7.9, and 2.3 Ci (4 mM) of [ 3 H]butanol.…”

mentioning

confidence: 99%

Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor

Hatton

Lintz

Molecular and Cellular Biology

et al. 2015

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Overexpression of epidermal growth factor receptor (EGFR) is one of the frequent mechanisms implicated in cancer progression, and so is the overexpression of the enzyme phospholipase D (PLD) and its reaction product, phosphatidic acid (PA). However, an understanding of how these signaling molecules interact at the level of gene expression is lacking. Catalytically active PLD enhanced expression of EGFR in human breast cancer cells. Overexpression of the PLD2 isoform increased EGFR mRNA and protein expression. It also negated an EGFR downregulation mediated by small interfering RNA targeting EGFR (siEGFR). Several mechanisms contributed to the alteration in EGFR expression. First was the stabilization of EGFR transcripts as PLD2 delayed mRNA decay, which prolonged their half-lives. Second, RNase enzymatic activity was inhibited by PA. Third, protein stabilization also occurred, as indicated by PLD resistance to cycloheximide-induced EGFR protein degradation. Fourth, PA inhibited lysosomal and proteasomal degradation of internalized EGFR. PLD2 and EGFR colocalized at the cell membrane, and JAK3 phosphorylation at Tyr980/Tyr981 followed receptor endocytosis. Further, the presence of PLD2 increased stabilization of intracellular EGFR in large recycling vesicles at ϳ15 min of EGF stimulation. Thus, PLD2-mediated production of PA contributed to the control of EGFR exposure to ligand through a multipronged transcriptional and posttranscriptional program during the out-of-control accumulation of EGFR signaling in cancer cells. Epidermal growth factor receptor (EGFR) is overexpressed in many epithelial tumors, including bladder, kidney, pancreatic, and squamous cell carcinomas (1). In breast cancer, EGFR overexpression is associated with advanced-stage disease and shortened relapse-free survival, which occurs concomitantly with low estrogen receptor expression (2). The cell signaling events after EGFR ligand stimulation and cancer have been greatly studied and require association of the receptor with a number of cytoplasmic tyrosine kinases, as well as activation of the Janus kinase (JAK)/ STAT pathway (3). EGFR directly interacts with phospholipase D2 (PLD2) (4-6). Stimulation of EGFR increases cellular PLD activity and the production of phosphatidic acid (PA) in cancer cell lines (7,8). PA is also the precursor to lysophosphatidic acid (LPA) that is relevant in ovarian cancer (9). PLD2 activity is regulated by the phosphorylation of the kinases EGFR and Janus kinase 3 (JAK3) on the Y296 and Y415 tyrosine residues, respectively (10, 11). JAK3 is a 130-kDa intracellular, nonreceptor tyrosine kinase (12, 13). It can also function as the docking site for other proteins if they have the proper Src homology 2 (SH2) domains.Tyrosine kinases associated with EGFR, such as Fer/Fes, promote cell motility in a PLD/PA-dependent pathway (14). We have recently found that Fes binds PA and participates in a PLD-induced pathway of myeloid differentiation (15). PLD2 is associated with EGFR signaling by binding to Grb2 at two specific res...

“…The PX domain of PLD2 contains the site for GEF activity, whereas both the PLD2-PX and -PH domains are involved in interacting with the small GTPase substrate (24), which can be either Rac2 or Ras (22,25). …”

Section: Introductionmentioning

confidence: 99%

Mechanism of enzymatic reaction and protein–protein interactions of PLD from a 3D structural model

Alter

Gómez‐Cambronero

2015

Cellular Signalling

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The phospholipase D (PLD) superfamily catalyzes the hydrolysis of cell membrane phospholipids generating the key intracellular lipid second messenger phosphatidic acid. However, there is not yet any resolved structure either from a crystallized protein or from NMR of any mammalian PLDs. We propose here a 3D model of the PLD2 by combining homology and ab initio 3 dimensional structural modeling methods, and docking conformation. This model is in agreement with the biochemical and physiological behavior of PLD in cells. For the lipase activity, the N- and C-terminal histidines of the HKD motifs (His 442/His 756) form a catalytic pocket, which accommodates phosphatidylcholine head group (but not phosphatidylethanolamine or phosphatidyl serine). The model explains the mechanism of the reaction catalysis, with nucleophilic attacks of His 442 and water, the latter aided by His 756. Further, the secondary structure regions superimposed with bacterial PLD crystal structure, which indicated an agreement structure model obtained. It also explains protein-protein interactions, such as PLD2-Rac2 transmodulation (with a 1:2 stoichiometry), PLD2 GEF activity on Rac2 that is relevant for actin polymerization and cell migration, and a biding site for phosphoinositides. Since tumor-aggravating properties have been found in mice overexpressing PLD2 enzyme, the 3D model of PLD2 will be also useful, to a large extent, in developing pharmaceuticals to modulate its in vivo activity.