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Phospholipase D2 (PLD2) Is a Guanine Nucleotide Exchange Factor (GEF) for the Gtpase Rac2

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Phospholipase D2 (PLD2) Is a Guanine Nucleotide Exchange Factor (GEF) for the Gtpase Rac2 Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2 Madhu Mahankalia, Hong-Juan Penga, Karen M. Henkelsa, Mary C. Dinauerb, and Julian Gomez-Cambroneroa,1 aDepartment of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435; and bDepartment of Pediatrics (Division of Hematology/Oncology) and Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110 Edited by Peter N. Devreotes, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 21, 2011 (received for review September 7, 2011) We have discovered that the enzyme phospholipase D2 (PLD2) binds known that PA binds directly to Sos (9). All of the mechanisms directly to the small GTPase Rac2, resulting in PLD2 functioning as a that imply translocation to the membrane or membrane traf- guanine nucleotide exchange factor (GEF), because it switches Rac2 ficking that place GEF close to its effectors are complex and rely from the GDP-bound to the GTP-bound states. This effect is large on the formation of multimeric signaling proteins. enough to be meaningful (∼72% decrease for GDP dissociation and We show here that PLD2 functions as a GEF, because it 300% increase for GTP association, both with PLD2), it has a half- switches Rac2 from the GDP-bound to the GTP-bound state. time of ∼7 min, is enhanced with increasing PLD2 concentrations, There are a number of known GEFs, but none of the three families and compares favorably with other known GEFs, such as Vav-1. The of GEFs for monomeric GTPases is a phospholipase, making this PLD2-Rac2 protein–protein interaction is sufficient for the GEF func- study unique. A further biological significance is that signals from tion, because it can be demonstrated in vitro with just recombinant the cell receptor are routed to Rac with an economy of molecular proteins without lipid substrates, and a catalytically inactive lipase machinery without any intermediary factor(s). Last, our results are (PLD2-K758R) has GEF activity. Apart from this function, exogenous demonstrated first in vitro and then extended to the physiology of phosphatidic acid by itself (300 pM) increases GTP binding and leukocytes, implicating PLD2 as a GEF for Rac in key cellular enhances PLD2-K758R–mediated GTP binding (by ∼34%) but not functions: cell adhesion, chemotaxis, and phagocytosis. GDP dissociation. Regarding the PLD2-Rac2 protein–protein associa- Results tion, it involves, for PLD2, residues 263–266 within a Cdc42/Rac in- CELL BIOLOGY teractive binding region in the PH domain, as well as the PX domain, Rac2 and PLD2 Interact in Vivo and in Vitro. Initially, we hypothe- and it involves, for Rac2, residue N17 within its Switch-1 region. sized that both PLD2 and Rac2 could form an interaction within A PLD2’s GEF function is demonstrated in living cells, because silencing the cell and must, therefore, be in close spatial proximity. Fig. 1 PLD2 results in reduced Rac2 activity, whereas PLD2-initiated Rac2 demonstrates that Rac2 and PLD2 are associated in the cell by fl activation enhances cell adhesion, chemotaxis, and phagocytosis. immuno uorescence microscopy. FITC-labeled Rac2 and tetra- There are several known GEFs, but we report that this GEF is har- methyl rhodamine isothiocyanate (TRITC)-labeled PLD2 coloc- bored in a phospholipase. The benefit to the cell is that PLD2 brings alize to the perinuclear region in the cell, though some Rac2 also spatially separated molecules together in a membrane environment, has a punctate nature localized to endosomes. To further study ready for fast intracellular signaling and cell function. the association of Rac2 and PLD2, and to ascertain the stoichi- ometry of the binding, we used recombinant proteins from a baculovirus/insect cell expression system, as our laboratory re- he small GTPase Rac is a crucial regulator of actin cytoskeletal cently documented (10). This process involved expression and Trearrangement and plays an important role in cell spreading, purification of C-terminal 6xHN-tagged proteins by a TALON migration, mitogenesis, phagocytosis, superoxide generation, and cobalt metal affinity resin. PLD2 was >87% pure (Fig. 1B), axonal growth (1). Activation of Rac occurs because of GTP ex- whereas Rac2 was >95% pure (Fig. 1C). To ascertain the stoi- change factors [guanine nucleotide exchange factors (GEFs)], and chiometry of PLD2 and Rac2 binding, we used these recombinant inactivation is mediated by the intrinsic GTPase enzymatic activity proteins in an ELISA binding assay. We added increasing con- of Rac, greatly aided by GTPase activating proteins (GAPs). centrations of PLD2 to the plate and determined the bound Further regulation of the cycle is mediated by guanosine nucleo- fraction. Then, we added a constant and excess amount of Rac2 tide dissociation inhibitors (GDIs) that antagonize both GEFs and and measured the molar concentration of Rac2 bound to the GAPs. There are three families of GEFs for monomeric GTPases PLD2 by direct UV absorbance (Fig. 1D). With this technique, we that include Cdc25, Dbl homology (DH), and Sec7 domains (2–4). calculated that PLD2 binds to Rac2 at a molar ratio of PLD2: Clear advances have been made in the molecular mechanisms Rac2 between 1:1.3 and 1:1.7. Results in Fig. 1 thus indicate that that control the targeting of GEFs to their effectors, such as Rac, Rac2 and PLD2 form a heterodimer. to the plasma membrane, but only a handful of cases remain clearly mapped out. For Sos, the recruitment of phosphatidyli- PLD2 Is a Bona Fide GEF for Rac2. To investigate the possible nositol 3,4,5 phosphate (PIP3) concurs to unmask its Rac-GEF functional consequences of a Rac2-PLD2 interaction on Rac2 activity in vitro (5). Actin-binding ezrin promotes recruitment of functionality, we designed a loss-of-function cell model in Rac2 GEF-Dbl to the plasma membrane and to lipid raft microdomains GTPase activity. For this, we used RNA gene expression silencing where it can act upon Cdc42 and the downstream effector PAK-1 and analyzed Rac2 activation [protein binding domain (PBD) (6). Also, activation of ARFs catalyzed by the characteristic Sec7 pull-down]. As shown in the Western blots depicted in Fig. 2A, domain of ARF-GEFs occurs before recruitment of their effec- tors and membrane trafficking and vesicle formation (7). Several studies have implicated phospholipase D (PLD) in cell Author contributions: J.G.-C. designed research; M.M., H.-J.P., and K.M.H. performed re- signaling, as it relates to Rac. The C-terminal polybasic motif of search; M.C.D. contributed new reagents/analytic tools; M.M., H.-J.P., K.M.H., M.C.D., and Rac1 interacts with phosphatidic acid (PA), and PLD serves to J.G.-C. analyzed data; and J.G.-C. wrote the paper. recruit Rac to the cell membrane (7), but how this affects the The authors declare no conflict of interest. GEFs for Rac is not known. However, PLD-derived PA acts upon This article is a PNAS Direct Submission. GEFs. PA activates the unconventional DOCK2-GEF, which 1To whom correspondence should be addressed. E-mail: [email protected]. then targets GTPase(s) (8). Our laboratory demonstrated that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PLD binds to Grb2, which recruits the GEF Sos (8), and it is also 1073/pnas.1114692108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114692108 PNAS | December 6, 2011 | vol. 108 | no. 49 | 19617–19622 Downloaded by guest on October 2, 2021 A had a positive effect on Rac2 activation, because its lack of ex- pression led to a diminished Rac2 activation. Conversely, si- lencing with siPLD1 RNA (Fig. 2B for immunoblot controls) or siScrambled RNA had no effect on Rac2 activity (Fig. 2C). This Rac2 PLD2 DAPI Merge in vivo study suggests a PLD2-led GEF effect on Rac2 (but not a PLD1-led effect) with functional consequences. Thus, we con- B kDa centrated on the PLD2 isoform for all subsequent experiments. Crude Flow ThruWash #1Wash #2Wash Elution#3 Elution #1 Elution #2 Elution#3 Elution#4 #5 223 The first evidence that PLD2 acted as a GEF for Rac2 is 109 PLD2 83 shown in Fig. 2 D–G and represents a series of GTP loading 47 1.0 experiments in the presence of increasing PLD2 concentrations. D PLD2 (Added 1st) 32 Recombinant Proteins Rac2 (Added 2nd) 26 In an in vivo approach shown in Fig. 2D, Sf21 cells were coin- 16 0.8 fected with fixed amounts of baculoviral Rac2 and varying 110 kDa PLD2 W.B. αmyc 0.6 amounts of baculoviral PLD2. Increasing expression of PLD2 led to an increase in GTP/GDP exchange loading activity of Rac2. C 0.4 kDa Crude Flow ThruWash #1Wash #2Wash Elution#3 Elution #1 Elution #2 #3 PLD2 had an overall positive effect on Rac2 activity at a multi- 223 Stoichiometry plicity of infection (MOI) ≥ 10:1. This effect was also observed in 109 0.2 83 (Absorbance at 280 nm) COS-7 cells transfected with PLD2 and/or Rac2 (Fig. 2E). 47 fi 0.0 The use of recombinant, puri ed proteins (Rac2 and PLD2) 32 0 0.5 1 1.5 2 2.5 26 Rac2 γ [Bound Protein] (μM) indicated that GTP S alone had a weak effect on GTP loading of 16 Rac2 at low nanomolar concentrations (Fig. 2F), which increased 26 Rac2 kDa concomitantly with increasing concentrations of recombinant W.B. αHA PLD2 (Fig. 2G). It is noteworthy that the results had no exog- Fig. 1. Demonstration of a molecular association between Rac2 and PLD2. (A) enous lipids (neither phosphatidylcholine, the substrate of PLD2 Immunofluorescence microscopy of COS-7 cells cotransfected with both myc- action, nor the product, PA), indicating that the effect of PLD2 Rac2 and HA-PLD2 alone.
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