Negative Charges in the Flexible N-Terminal Domain of Rho GDP-Dissociation Inhibitors (RhoGDIs) Regulate the Targeting of the RhoGDI−Rac1 Complex to Membranes This information is current as of September 24, 2021. Takehiko Ueyama, Jeonghyun Son, Takeshi Kobayashi, Takeshi Hamada, Takashi Nakamura, Hirofumi Sakaguchi, Toshihiko Shirafuji and Naoaki Saito J Immunol 2013; 191:2560-2569; Prepublished online 5 August 2013; Downloaded from doi: 10.4049/jimmunol.1300209 http://www.jimmunol.org/content/191/5/2560 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/08/06/jimmunol.130020 Material 9.DC1 References This article cites 33 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/191/5/2560.full#ref-list-1

Why The JI? Submit online. by guest on September 24, 2021

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Negative Charges in the Flexible N-Terminal Domain of Rho GDP-Dissociation Inhibitors (RhoGDIs) Regulate the Targeting of the RhoGDI–Rac1 Complex to Membranes

Takehiko Ueyama,* Jeonghyun Son,* Takeshi Kobayashi,† Takeshi Hamada,* Takashi Nakamura,* Hirofumi Sakaguchi,‡ Toshihiko Shirafuji,* and Naoaki Saito*

In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) a forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDIa from the RhoGDIa–Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI–Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI–Rac complex, during FcgR-

mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDIa, particularly its second Downloaded from negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI–Rac complex. We also found that RhoGDIa translocated to the phagosomes as a RhoGDIa–Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDIa–Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDIa, 2) the binding affinity of RhoGDIa for Rac1, and 3) anionic phospho- http://www.jimmunol.org/ lipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDIa–Rac1 complex is also applicable for the RhoGDIb-Rac1 complex. The Journal of Immunology, 2013, 191: 2560–2569.

mall are cell signaling molecules switching be- teins (3) and p75NTR (4); (PAK1, Src, and tween active (GTP-bound) and inactive (GDP-bound) forms. Cs) that phosphorylate RhoGDI (1, 5, 6); and phospholipids The cycles of Rho-family and -family small GTPases (1,2,7). S phox phox (RhoGTPases and RabGTPases) are regulated by three major Rac is one of the four cytoplasmic activators (p47 , p40 , phox factors: guanine nucleotide exchange factors, GTPase-activating p67 , and Rac) of the phagocyte (Nox2-based) NADPH oxi- by guest on September 24, 2021 , and GDIs (1, 2). The inactive forms of RhoGTPases dase, which produces reactive oxygen species (ROS) in response are sequestered in the cytoplasm by dimerization with Rho GDP- to various receptor-mediated signaling events (8). These four ac- dissociation inhibitors (RhoGDIs). During their activation, they tivators are further categorized as two protein complexes: a Rac are released from RhoGDIs to be converted into the GTP-bound complex (Rac–RhoGDI) and PHOX complex (p47phox-p67phox- form to execute a specific function on membranes (1, 2). The p40phox). In resting states, the two protein complexes are inactive dissociation of RhoGDI from Rac is further regulated by three in the cytoplasm. During cell activation, the multiprotein Nox2- major factors, known as the RhoGDI dissociation factors (1, 2): p22phox-p47phox-p40phox-p67phox-Rac is formed by independent RhoGDI-displacement proteins, such as the ERM family pro- translocation of the two complexes to membrane-spanning Nox2- p22phox (8). Reviews focusing on phagocytes (9, 10) have specu- *Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe Univer- lated that the dissociation of Rac from RhoGDI occurs in the cy- sity, Kobe 657-8501, Japan; †Department of Physiology, Nagoya University Graduate toplasm. However, no systematic study currently exists that clarifies ‡ School of Medicine, Nagoya 466-8550, Japan; and Department of Otolaryngology– this mechanism. Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto 606-8507, Japan The family of RhoGDIs contains three isoforms: RhoGDIa, Received for publication January 23, 2013. Accepted for publication July 1, 2013. RhoGDIb (LyGDI), and RhoGDIg. RhoGDIa is ubiquitously This work was supported in part by Grants-in-Aid for Scientific Research on Inno- expressed, whereas RhoGDIb is predominantly expressed in he- vative Areas for “Fluorescence Live Imaging” from the Ministry of Education, Cul- matopoietic cells, and RhoGDIg is expressed primarily in the ture, Sports, Science and Technology, Japan, as well as by a Japan–Hungary Research brain. In resting cells, RhoGDIa and RhoGDIb are localized to Cooperative Program grant from the Japan Society for the Promotion of Science and the Hungarian Academy of Sciences. the cytoplasm with RhoGTPases (1). In sharp contrast, RhoGDIg is Address correspondence and reprint requests to Dr. Takehiko Ueyama and Dr. Naoaki localized at the Golgi apparatus through its unique amphipathic N- Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe terminal segment (aa 1–33) (11). The structure of isolated RhoGDIa/ University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. E-mail addresses: b comprises two distinct regions: an N-terminal unstructured arm [email protected] (T.U.) and [email protected] (N.S.) (aa 1–58 in RhoGDIa, aa 1–55 in RhoGDIb), and an Ig-like folded The online version of this article contains supplemental material. domain (aa 59–204 in RhoGDIa, aa 56–201 in RhoGDIb) con- Abbreviations used in this article: DGK, diacylglycerol kinase; GDI, GDP- dissociation inhibitor; mKO, monomeric Kusabira orange; PA, phosphatidic acid; taining a hydrophobic pocket that packs the isoprenylated C-terminal pAb, polyclonal Ab; PB, polybasic; PM, plasma membrane; RhoGDI, Rho GDP- tail of Rac (12). The N termini of RhoGDIa (25 residues) and dissociation inhibitor; RhoGTPase, Rho-family small GTPase; ROS, reactive oxygen RhoGDIb (22 residues) possess numerous negatively charged amino species. acids: 8 in RhoGDIa and10inRhoGDIb. Based on structural Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 studies of the RhoGDIa–Rac1 (12, 13) and RhoGDIb–Rac2 (14) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300209 The Journal of Immunology 2561 complexes, the 25 and 22 N-terminal residues were found to re- hours after the transfection, the cells were lysed in 250 ml in lysis buffer main flexible even in the RhoGDI–Rac complex. (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, With respect to the translocation of Rac to the phagosomes, other 0.25% Triton X-100) by sonication. The total cell lysates were centrifuged at 12,000 3 g for 20 min at 4˚C, and the supernatants were incubated with researchers and we (15, 16) have reported an interaction between the 20 ml c-Myc(9E10) mAb–conjugated agarose (or 10 ml HA(TANA2) polybasic (PB) motif in the C terminus of Rac (K183KRKRK188)and mAb–conjugated magnetic agarose) for 2 h at 4˚C. The precipitates were anionic phospholipids, such as phosphatidic acid (PA) and phos- washed three times, and the aliquots of the precipitate were subjected to SDS- phatidylserine, is a key determinant. Previous studies established the PAGE (12.5%) followed by immunoblotting using a GFP pAb (1:1000). The bound Abs were detected with secondary Ab-HRP conjugates using the ECL identification of RhoGDIa as a component of the phagosome pro- detection system (GE Healthcare). teasome (17), the accumulation of RhoGDI on phagosomes (18), and phosphorylation of RhoGDIa (5, 19) by phagosome-targeting kina- Confocal fluorescence imaging of fixed cells or live cells ses (18, 20). Based on these observations, we hypothesized that the RAW246.7 cells were seeded onto 35-mm glass-bottom dishes (MatTek RhoGDIa–Rac complex translocates to phagosomes, and then Rac is chambers) and transfected using 2.53 volume of FuGENE HD reagent dissociated/activated on the phagosomes. Moreover, we hypothesized (Promega). After 48 h, cells were stimulated with BIgG and fixed 5 min that the negatively charged and flexible N terminus (25 residues) of later with 4% paraformaldehyde in 0.1 M PB buffer for 30 min at room RhoGDIa functions as a suppressor for the phagosomal recruitment termperature. The fixed cells were permeabilized using PBS containing 0.3% Triton X-100 for 10 min and stained using IgG primary Ab (aHA: of the RhoGDIa–Rac complex. In this study, we verified our hypoth- 1:250, aRac1: 1:500, 2 h at room temperature). The primary Abs were eses, and they are applicable not only for RhoGDIa but also for visualized on a confocal laser-scanning fluorescence microscope (LSM RhoGDIb in FcgR-mediated phagocytosis. 700; Carl Zeiss) using Alexa-conjugated anti-IgG (Invitrogen; 1:2000, 0.5 h

at room temperature). For live imaging 45–48 h after the transfection, the Downloaded from culture medium was replaced with HBSS(+) (Wako). After BIgG was Materials and Methods added to each plate, the images were collected at 5-s intervals for 10 min using a confocal laser-scanning fluorescence microscope with a heated Materials objective. All imaging experiments were performed in triplicate or more The polyclonal Ab (pAb) against RhoGDIa, mAb against c-Myc(9E10)– for at least three independent transfection experiments (n $ 9). conjugated agarose resin, and mAb against GAPDH were purchased from Santa Cruz Biotechnology. The mAb against HA(TANA2)-conjugated ROS production assay http://www.jimmunol.org/ magnetic agarose were from MBL International. The mAbs against HA HEK293Nox2/FcgRIIa cells in six-well dishes were transfected with various (3F10) and Myc(9E10) were from Roche. The mAb against Rac1 was from phox phox phox combinations of plasmids using FuGENE6 40–48 h before the assay. The Millipore. The pAbs against p47 , p67 , and p40 were described ROS release in response to BIgG from 2.0 3 105 trypsinized cells was previously (21). The rabbit pAb against GFP was made in-house. The IgG- measured in HBSS(+) during 15 min by a luminol-enhanced chemilumi- opsonized 2-mm glass beads (BIgG; Duke Scientific) were prepared as nescence method using a luminometer (Mithras LB940; Berthold) (22). described previously (20). Consistency in protein expression was confirmed by immunoblotting of total lysate for the same number of cells. Cell culture Statistical analysis The RAW264.7 macrophages (15) and HEK293 line with stable expres- Nox2/FcgRIIa sion of Nox2 and FcgRIIa (HEK293 ) (22) were maintained in The production of ROS is presented as a percentage relative to the control by guest on September 24, 2021 DMEM (Wako Pure Chemical Industries) containing 10% FBS (Invitrogen) experiment. The phagosomal accumulation of proteins is expressed using and in Eagle’s MEM (Wako) containing 10% FBS and 100 mM nones- the ratio of fluorescence intensity (phagosome/cytoplasm). Immunopreci- sential amino acids (Wako). For establishing clonally derived HEK293 cell pitated protein bands were quantified using ImageJ (National Institutes line with stable knockdown of Rac1 (HEK293Rac1KD), pSUPER-Rac1 of Health) and is expressed as a ratio relative to RhoGDIa. All data are (618) (23) was transfected into HEK293 cells using FuGENE6, followed presented as the means 6 SEM. Significant differences between two by clone selection in the presence of 1 mg/ml G418 (Wako). groups (p , 0.05) were identified using an unpaired two-tailed Student t test by Prism 5.0 (GraphPad Software). Construction of plasmids Human RhoGDIa, RhoGDIb, and RhoGDIg were amplified by PCR using Results first-strand cDNA (BD Biosciences), cloned into the pEGFP(N1) vector Three isoforms of RhoGDI (Invitrogen), and named RhoGDIa-GFP, RhoGDIb-GFP, and RhoGDIg-GFP. They were also cloned into the phmKO1(MN1) vector (humanized mono- The three RhoGDI isoforms were expressed in RAW264.7 cells, with meric Kusabira orange; excitation, 548 nm, emission, 561 nm; Amalgaam) mRNA levels significantly higher for RhoGDIa and RhoGDIb than (24) and named RhoGDIa-mKO, RhoGDIb-mKO, and RhoGDIg-mKO. The those for RhoGDIg (Supplemental Fig. 1A). Immunolocalization in phox phox phox expression plasmids of p47 ,p67 ,p40 (22), Myc-Rac1 (23), GFP- RAW264.7 cells revealed that RhoGDIa-mKO, whose tag is a red Rac1, and GFP-Rac1(6A) (15), whose PB motif in the C terminus is altered fluorescent protein named as monomeric Kusabira orange with by six Ala and is a phagosome-targeting defective mutant (15), were de- scribed previously. Mouse HA-PLD2 was a gift from Dr. Frohman (25, 26). excitation of 548 nm and emission of 561 nm (24), and RhoGDIb- Catalytically inactive PLD2 with the H442D (25) was constructed mKO were localized in the cytoplasm, whereas RhoGDIg-mKO using the QuikChange XL II mutagenesis kit (Stratagene). Various mutants, was confined to the Golgi apparatus (data not shown). In contrast, including RhoGDIa(D45A), RhoGDIa(D185A), RhoGDIa(D45/185A), GFP-Rac1 was localized to the plasma membrane (PM), in ad- RhoGDIa(3A), RhoGDIa(5A), RhoGDIa(8A), RhoGDIa(8A:D45/185A), RhoGDIb(D42A), and RhoGDIb(D182A) both in pEGFP(N1) and phmKO dition to the cytoplasm and nucleus (Supplemental Fig. 1B). When (MN1), were generated using QuikChange. N-terminal deletion mutants, coexpressed with RhoGDIs, GFP-Rac1 was not detected at the PM including DΝ15-RhoGDIa, DΝ25-RhoGDIa,andDΝ22-RhoGDIb both in but was colocalized with RhoGDIa-mKO and RhoGDIb-mKO in pEGFP(N1) and phmKO(MN1), were constructed using PCR. The short hairpin the cytoplasm and with RhoGDIg-mKO in the Golgi (Supple- RNA expression vectors pSUPER(neo) and pSUPER(gfp-neo) (Oligoengine) mental Fig. 1B). These data indicated that the subcellular locali- containing a Rac1-specific knockdown target sequence (nucleotides 618–636 from ATG), pSUPER-Rac1(618), and pSUPER-Rac1(618)gfp were de- zation of Rac1 is regulated by RhoGDIs. scribed previously (23). Translocation of the RhoGDIa–Rac1 complex to phagosomes Immunoprecipitation and immunoblotting Because RhoGDIa was reported to accumulate on phagosomes of The Myc-Rac1 and RhoGDI-GFP constructs (HA-PLD2 plus GFP-Rac1, or J744 macrophages (detected by proteomics) and primary neutrophils HA-PLD2 plus RhoGDIa-GFP) were cotransfected into HEK293 cells (detected by immunocytochemistry) during FcgR-mediated phago- plated on 10-cm dishes using 2.53 volume of FuGENE6. Forty-eight cytosis (17, 18), we hypothesized that the RhoGDIa–Rac1 complex 2562 NEGATIVELY CHARGED FLEXIBLE N TERMINI OF RhoGDIa/b migrates to the phagosome where it releases Rac in proximity to with Rac1 (Fig. 1F), which is consistent with a previous report Nox2-based NADPH oxidase. Fig. 1A shows that GFP-Rac1 accu- (27). Thus, these results suggested that RhoGDIa and Rac1 ac- mulated on the phagosomes during FcgR-mediated phagocytosis in cumulate on phagosomes as a complex during FcgR-mediated RAW264.7 cells, as shown previously (15). However, RhoGDIa- phagocytosis using the interaction between RhoGDIa and Rac1 mKO did not accumulate on the phagosomes (Fig. 1B). Moreover, after conformational changes in the complex. coexpression of RhoGDIa-mKO and GFP-Rac1 in RAW264.7 cells Next, we examined the effects of RhoGDIa, RhoGDIa(D45A), and did not lead to their accumulation on phagosomes (Fig. 1C). Our RhoGDIa(D185A) on ROS production using HEK293Nox2/FcgRIIa understanding of the discrepancy about accumulation of RhoGDIa cells. Untagged and C-terminally GFP- or mKO-tagged RhoGDIa between previous reports and the current study at that time was that constructs inhibited ROS production to a similar extent (data not detection of accumulated RhoGDIa on phagosomes is influenced shown). RhoGDIa-GFP completely inhibited ROS production by various factors, such as cell type used (primary or immortalized (1.2 6 0.2%), whereas RhoGDIa(D45A)-GFP and RhoGDIa cells, macrophages, or neutrophils) and detection method used (D185A)-GFP partially suppressed ROS production (62.7 6 7.0 (proteomics, immunocytochemistry, or confocal fluorescence imag- and 65.1 6 12.9%, respectively) (Fig. 1G). These data indicated ing). Then, we tested RhoGDIa-mKO mutants, RhoGDIa(D45A) that the inhibitory effect of RhoGDIa on FcgR-mediated ROS and RhoGDIa(D185A), characterized by weakened interactions production depends on its binding affinity to Rac1. with RhoGTPases due to partial conformational changes in RhoGDI– a RhoGTPase complexes (13, 14, 27). Neither mutant expressed in Negatively charged N terminus of RhoGDI suppresses RAW264.7 cells showed any accumulation on the phagosomes Rac-dependent membrane (PM and phagosome) targeting of a during FcgR-mediated phagocytosis (Supplemental Fig. 2). RhoGDI Downloaded from However, they both accumulated on phagosomes during FcgR- We hypothesized that the negatively charged flexible N terminus mediated phagocytosis when coexpressed with GFP-Rac1 (Fig. (25 residues) of RhoGDIa plays an important role in the subcellular 1D, 1E). Immunoprecipitation analysis showed that RhoGDIa localization of the RhoGDIa–Rac1 complex. First, we constructed (D45A) and RhoGDIa(D185A) maintained a weak interaction N-terminal deletion mutants of RhoGDIa: DN15-RhoGDIa-mKO http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Impact of RhoGDIs on Rac1 translocation to the phagosomes during phagocytosis and on ROS production. (A–E) GFP-Rac1 was cotransfected with mKO (A), RhoGDIa-mKO (C), RhoGDIa(D45A)-mKO (D), or RhoGDIa(D185A)-mKO (E) into RAW264.7 macrophages, and RhoGDIa-mKO was cotransfected with GFP (B). The transfected cells were stimulated with 2-mm BIgG and visualized under a confocal microscope. GFP- Rac1 (A) but not RhoGDIa-mKO (B) accumulates on phagosomes. Neither GFP-Rac1 or RhoGDIa-mKO accumulates on phagosomes (C). RhoGDIa (D45A)-mKO (D) and RhoGDIa(D185A)-mKO (E) coexpressed with GFP-Rac1 accumulate on phagosomes. The arrows and arrowheads indicate phagosomes engulfing BIgG. (F) Representative immunoprecipitation data (n $ 3) for the interaction between Myc-Rac1 and RhoGDIa-GFP or its mu- tants (D45A and D185A). RhoGDIa(D45A) and RhoGDIa(D185A) maintain a weak interaction with Rac1. (G) ROS production from HEK293Nox2/FcgRIIa cells transfected with a mock vector, RhoGDIa-GFP, RhoGDIa(D45A)-GFP, or RhoGDIa(D185A)-GFP in combination with Phox proteins and Myc-Rac1. The cells were stimulated with 2-mm BIgG, and ROS release was measured by luminol-enhanced chemiluminescence (n $ 5). RhoGDIa completely inhibited ROS production, whereas RhoGDIa(D45A) and RhoGDIa(D185A) partially suppressed ROS production. The comparable expression of the proteins was confirmed with immunoblotting. The Journal of Immunology 2563 and DN25-RhoGDIa-mKO (Fig. 2A). In resting RAW264.7 cells, Suppression of the Rac-dependent accumulation of the both mutants were detected in the cytoplasm (Fig. 2B, 2C), but RhoGDIa–Rac1 complex on phagosomes by negative charges DN25-RhoGDIa-mKO also exhibited a weak PM localization (Fig. in the N terminus of RhoGDIa 2C). During FcgR-mediated phagocytosis, only DN25-RhoGDIa- To further investigate the impact of the negative charges located in mKO was found accumulating on phagosomes (Fig. 2C). The PM the N terminus (25 residues) of RhoGDIa, we constructed three localization of DN25-RhoGDIa-mKO was also observed in mutants with reduced numbers of negatively charged amino acids: HEK293 cells (Fig. 3A). It disappeared in HEK293 cells with Rac1KD RhoGDIa(3A)-mKO, RhoGDIa(5A)-mKO, and RhoGDIa(8A)- stable knockdown of Rac1, HEK293 cells (Fig. 3B, 3C) and mKO (Fig. 4A). Immunoprecipitation analysis showed that all was restored by introduction of full-length GFP-Rac1 (nucleotides mutants bind Rac1 similar to RhoGDIa, contrary to a faint binding 1–598 from ATG), which is resistant to pSUPER-Rac1(618), into Rac1KD of Rac1 interaction-impaired RhoGDIa(8A:D45/185A) (Fig. 4B). HEK293 cells (Fig. 3D). Rac1-dependent PM localization/ Confocal microscopy revealed that the mutants RhoGDIa(3A)- recruitment of DN25-RhoGDIa-mKO was confirmed by single- mKO and RhoGDIa(5A)-mKO were primarily located in the cy- molecule imaging of tetramethylrhodamine-conjugated Halo-tagged toplasm of resting RAW264.7 cells, and they did not accumulate DN25-RhoGDIa (HaloTag;Promega)inHeLacellsbasedonres- on phagosomes during phagocytosis (Fig. 4C, 4D). In contrast, idence time, frequency of recruitment, and trajectory in PM: the RhoGDIa(8A)-mKO mutant exhibited remarkable PM lo- residencetimeandrecruitment in PM of DN25-RhoGDIa-Halo calization in resting RAW264.7 cells and accumulated on phago- (tetramethylrhodamine) were significantly extended and increased, somes during phagocytosis (Fig. 4E), contrary to cytoplasmic respectively, by coexpression of GFP-Rac1 (Supplemental Fig. 3). RhoGDIa(8A:D45/185A) (Fig. 4F). Endogenous Rac1 accumu- Immunoprecipitation analysis demonstrated that DN15-RhoGDIa lated with RhoGDIa(8A)-mKO on phagosomes (Supplemental Downloaded from and DN25-RhoGDIa had strong interactions with Rac1 similar Fig. 4A). These data suggested that membrane (PM and phago- to RhoGDIa (Fig.3E).FcgR-mediated ROS production in Nox2/FcgRIIa some) localization of RhoGDIa(8A)-mKO is mediated by its HEK293 cells was completely inhibited by DN25- binding to Rac1. The coexpression of GFP-Rac1 with RhoGDIa RhoGDIa-GFP or DN15-RhoGDIa-GFP, similar to RhoGDIa (5A)-mKO or RhoGDIa(3A)-mKO stimulated the phagosomal (Fig. 3F). These data suggested the following: 1) the negatively accumulation of RhoGDIa(5A)-mKO but not RhoGDIa(3A)- charged N terminus of RhoGDIa, particularly the sequence http://www.jimmunol.org/ 17 22 mKO (Supplemental Fig. 4B, 4C). The phagosomal accumula- E NEEDE , plays a pivotal role in retaining RhoGDIa in cytoplasm; tion of RhoGDIa(8A)-mKO with GFP-Rac1 disappeared when 2) the membrane (PM and phagosome) localization of RhoGDIa is GFP-Rac1(6A), a phagosome-targeting defective mutant (15), dependent on Rac1; and 3) the inhibitory effect of RhoGDIa on was coexpressed with RhoGDIa(8A)-mKO (Supplemental Fig. ROS production is probably determined by its binding affinity to 4D, 4E). Because RAW264.7 macrophages express all Rac iso- Rac1, but not on its localization. forms (15), we knocked down Rac1 in HEK293Nox2/FcgRIIa cells, in which Rac1 is a predominant Rac isoform, using pSUPER- Rac1(618)gfp (23). The phagosomal accumulation of RhoGDIa (8A)-mKO in HEK293Nox2/FcgRIIa cells (Fig. 4G) were signifi- by guest on September 24, 2021 cantly reduced when Rac1 was knocked down (Fig. 3H, 3I). Moreover, ROS production from HEK293Nox2/FcgRIIa cells was completely suppressed by RhoGDIa-GFP, RhoGDIa(3A)-GFP, RhoGDIa(5A)-GFP, and RhoGDIa(8A)-GFP. However, RhoGDIa (8A:D45/185A)-GFP caused only a mild suppression (65.9 6 9.6%) (Fig. 3J). Based on these results, we concluded that all eight, particularly five in the sequence of E17NEEDE22, negatively charged amino acids in the flexible N terminus of RhoGDIa function as a suppressor for the accumulation of the RhoGDIa– Rac1 complex on phagosomes and that the inhibitory effect by RhoGDIa on ROS production is mediated by the binding affinity of RhoGDIa to Rac1. PLD2 promotes the translocation of the RhoGDIa–Rac1 complex to phagosomes and enhances ROS production The interaction between the PB motif in the C terminus of Rac1 (K183KRKRK188) and PA is important for Rac1 accumulation on the phagosomes and for ROS production during FcgR-mediated phagocytosis (15); besides, we reported the accumulation of the PA- producing enzyme PLD2 on phagosomes (26). Coexpression of PLD2, but not the catalytically inactive mutant PLD2(H442D), in- duced the translocation of RhoGDIa-mKO and GFP-Rac1 to the FIGURE 2. Membrane (PM and phagosome) localization of RhoGDIa phagosomes of RAW264.7 cells (Fig. 5A, 5B). This response by is dependent on its negatively charged N terminus (25 aa). (A) The amino PLD2 disappeared when GFP-Rac1 was replaced by the phago- acid sequence of the N termini in human wild-type (WT) and mutant some-targeting defective mutant GFP-Rac1(6A), despite PLD2 ac- RhoGDIa (DN15, DN25). A red number in parenthesis indicates the total cumulation on phagosomes (Fig. 5C). No interaction between PLD2 number of negatively charged amino acids in their N termini. (B and C) B C and Rac1 or RhoGDIa was confirmed by immunoprecipitation DN15-RhoGDIa-mKO ( )orDN25-RhoGDIa-mKO ( ) was transfected Nox2/FcgRIIa into RAW264.7 cells. The transfected cells were stimulated with 2-mm analysis (Fig. 5D). Transfection of PLD2 in HEK293 BIgG. DN25-RhoGDIa-mKO but not DN15-RhoGDIa-mKO shows a cells increased FcgR-mediated ROS production by ∼3.5-fold weak PM localization and accumulation on phagosomes. Arrows and ar- (340.5 6 40.1%), whereas the PLD2(H442D) mutant did not en- rowheads indicate PM areas and phagosomes engulfing BIgG, respectively. hance ROS production (70.2 6 7.4%) (Fig. 5E). These data indi- 2564 NEGATIVELY CHARGED FLEXIBLE N TERMINI OF RhoGDIa/b Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 3. Membrane localization of RhoGDIa and its inhibitory effect on ROS production are dependent on Rac1. (A) GFP and DN25-RhoGDIa- mKO were cotransfected in HEK293 cells. DN25-RhoGDIa-mKO is localized at PM (arrows). (B) Stable knockdown of Rac1 in HEK293Rac1KD cells was confirmed by immunoblotting. (C and D) GFP plus DN25-RhoGDIa-mKO (C) or GFP-Rac1 plus DN25-RhoGDIa-mKO (D) was cotransfected into HEK293Rac1KD cells. The PM localization of DN25-RhoGDIa-mKO disappeared (C) and was restored by GFP-Rac1 (D). (E) Representative imunopre- cipitation data (n $ 3) showing strong interaction between Myc-Rac1 and DN15-RhoGDIa-GFP or DN25-RhoGDIa-GFP, similar to WT RhoGDIa.(F) HEK293Nox2/FcgRIIa cells were transfected with a mock vector, RhoGDIa-GFP, DN15-RhoGDIa-GFP, or DN25-RhoGDIa-GFP in combination with Phox proteins and Myc-Rac1. The cells were stimulated with BIgG, and ROS release was measured (n $ 5). DN15-RhoGDIa and DN25-RhoGDIa as well as RhoGDIa completely inhibited ROS production. The comparable expression of proteins was confirmed by immunoblotting. Scale bars, 10 mm. cated that RhoGDIa accumulates on phagosomes as a RhoGDIa– previous report on RhoGDIb showing a 10- to 20-fold lower affinity Rac1 complex, and the accumulation is mediated by interaction for Cdc42 (28). Although DN22-RhoGDIb maintained an interaction between the PB motif of Rac1 and the anionic phospholipid PA with Rac1, RhoGDIb(D42A) demonstrated no interaction (Fig. produced on phagosomes, that is, by a phagosome-targeting mech- 6E). The ROS production assay in HEK293Nox2/FcgRIIa cells anism of Rac1. revealed a moderate suppression by RhoGDIb (32.2 6 6.0%) or DN22-RhoGDIb (35.8 6 5.5%) and a faint suppression by b Negatively charged N terminus of RhoGDI suppresses the RhoGDIb(D42A) (92.1 6 4.5%) or RhoGDIb(D182A) (85.0 6 b Rac-dependent phagosomal targeting of RhoGDI 7.81%), which is a homologous mutant of RhoGDIa(D185A), in Finally, we tested the impact of the negatively charged N terminus contrast to the complete suppression by RhoGDIa (Fig. 6F). There (22 residues) of RhoGDIb (Fig. 6A) on phagocytosis and ROS was no statistical difference between RhoGDIb and DN22-RhoGDIb production. When GFP-Rac1 was coexpressed with RhoGDIb-mKO with regard to binding affinity for Rac1 or inhibitory effect on ROS in RAW264.7 cells, GFP-Rac1 (but not RhoGDIb-mKO) accumu- production. lated on phagosomes during FcgR-mediated phagocytosis (Fig. 6B), in sharp contrast with GFP-Rac1 plus RhoGDIa-mKO (Fig. 1C). Discussion Even RhoGDIb(D42A)-mKO, which is a homologous mutant of The negatively charged and flexible N-terminal domain of RhoGDIa RhoGDIa(D45A), did not accumulate on phagosomes, despite sig- (25 residues) and RhoGDIb (22 residues) contains two highly nificant GFP-Rac1 accumulation (Fig. 6C). However, deletion of the conserved clusters of negatively charged amino acids (Fig. 7A). The Nterminus(DN22-RhoGDIb-mKO) allowed RhoGDIb to accumu- first cluster consists of three and two negative amino acids in human late with GFP-Rac1 on phagosomes (Fig. 6D). Immunoprecipitation RhoGDIa and RhoGDIb, respectively. The second cluster consists analysis indicated that RhoGDIb has a much weaker interaction with of five and eight negative amino acids in human RhoGDIa and Rac1 than RhoGDIa (Fig. 6E). This finding is consistent with a RhoGDIb, respectively. In the present study, we demonstrated that The Journal of Immunology 2565 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Negatively charged amino acids in the N terminus of RhoGDIa suppress Rac1-dependent translocation of the RhoGDIa–Rac1 complex to phagosomes. (A) Amino acid sequence of the N termini in human wild-type (WT) and mutant RhoGDIa (3A, 5A, and 8A). A red number in parenthesis indicates the total number of negatively charged amino acids in their N termini. The blue number and blue A indicate the total number and position of mutated Ala, respectively. (B) Representative immunoprecipitation data (n $ 5) showing strong interaction between Myc-Rac1 and RhoGDIa-GFP (WT, 3A, 5A, 8A) and a faint interaction between Myc-Rac1 and RhoGDIa(8A:D45/185A)-GFP. (C–F) RhoGDIa(3A)-mKO (C), RhoGDIa(5A)-mKO (D), RhoGDIa(8A)-mKO (E), or RhoGDIa(8A,D45/185A)-mKO (F) was transfected into RAW264.7 cells. The transfected cells were stimulated with 2-mm BIgG. RhoGDIa(8A)-mKO (E), but not RhoGDIa(8A:D45/185A)-mKO (F), shows PM localization and accumulates on phagosomes. The arrow and arrowheads indicate PM and phagosomes engulfing BIgG, respectively. (G and H) RhoGDIa-mKO(8A) was cotransfected with pSUPER(gfp-neo) (G)or pSUPER-Rac1(681)gfp (H) in HEK293Nox2/FcgRIIa cells. Cotransfection of pSuper-Rac1(681)gfp was confirmed by the expression of GFP (small panel in middle). The arrowheads indicate the phagosomes engulfing BIgG. Right graphs show fluorescence intensity profile of RhoGDIa(8A)-mKO detected along the arrows. (I) Quantification of fluorescence intensity ratio (phagosome/cytoplasm) of RhoGDIa(8A)-mKO in (G) and (H). Studies included n $ 30 from at least four individual experiments ($12 dishes; *p , 0.01). (J) ROS production from HEK293Nox2/FcgRIIa cells transfected with a mock vector, RhoGDIa- GFP, RhoGDIa(3A)-GFP, RhoGDIa(5A)-GFP, RhoGDIa(8A)-GFP, or RhoGDIa(8A,D45/185A)-GFP in combination with Phox proteins and Myc-Rac1. The cells were stimulated with BIgG, and ROS release was measured (n $ 5). RhoGDIa(8A) shows complete suppression, but RhoGDIa(8A:D45/185A) shows only a mild suppression. The comparable expression of proteins was confirmed by immunoblotting. all eight N-terminal negative amino acids of RhoGDIa (particularly the isoprenylated tail at C-terminal Cys189 position, which interacts five in the second cluster) function as a suppressor for the membrane with a hydrophobic pocket of RhoGDIa (12, 13), and 2) the PB targeting of the RhoGDIa–Rac1 complex and for RhoGDIa itself. motif (K183KRKRK188 in Rac1, R183QQKRA188 in Rac2) next to the The Rac proteins possess two membrane-targeting motifs (15): 1) isoprenylated tail. Whereas the C-terminal of Rac1 encompassing 2566 NEGATIVELY CHARGED FLEXIBLE N TERMINI OF RhoGDIa/b Downloaded from http://www.jimmunol.org/

FIGURE 5. PA produced by PLD2 promotes the translocation of the RhoGDIa–Rac1 complex to phagosomes and enhances ROS production. (A–C) RhoGDIa-mKO was cotransfected with GFP-Rac1 and HA-PLD2 (A), GFP-Rac1 and HA-PLD2(H442D) (B), or GFP-Rac1(6A) and HA-PLD2 (C) into RAW264.7 macrophages. The transfected cells were stimulated with 2-mm BIgG. After 5 min, fixed cells were stained with a hemagglutinin (HA) mAb and visualized with a confocal microscope using an Alexa647-conjugated anti-IgG. Accumulated PLD2 (A), but not PLD2(H442D) (B), induces the trans- by guest on September 24, 2021 location of RhoGDIa-mKO and GFP-Rac1 to phagosomes. Accumulated PLD2 induces no translocation of RhoGDIa-mKO when GFP-Rac1(6A), a phagosome-targeting defective mutant of GFP-Rac1, was used. The arrows, arrowheads, and double arrows indicate phagosomes engulfing BIgG. Scale bar, 10 mm. (D) Representative immunoprecipitation data (n $ 3) showing no interaction between HA-PLD2 and GFP-Rac1 or RhoGDIa-GFP. (E) HEK293Nox2/FcgRIIa cells were transfected with a mock vector, HA-PLD2 or HA-PLD2(H442D), in combination with Phox proteins. The cells were stimulated with BIgG, and ROS release was measured (n $ 5). PLD2 but not PLD2(H442D) enhances ROS production. The comparable expression of proteins was confirmed by immunoblotting.

residues 180–189 (Rac2 encompassing residues 182–189), but not the constant presence of phosphatidylserine, an anionic phospholipid, the isoprenylated tail, is poorly defined in the crystal structure of the on phagosomes and its significant contribution to targeting and re- RhoGDIa–Rac1 (RhoGDIb–Rac2) complex, the negatively charged taining proteins containing a PB cluster (16). In the present study, N-terminal of RhoGDIa/b and the PB motif in C-terminal of Rac1/2 we demonstrated that RhoGDIa translocates to phagosomes as a are expected to be in close proximity around the exit of the hydro- RhoGDIa–Rac1 complex through the phagosome-targeting mecha- phobic pocket of RhoGDIa/b (13, 14). It was reported that a peptide nism of Rac1 (Rac1-dependent mechanism) by the following ex- of Glu5-Glu20 from human RhoGDIa, which contains five negatively periments: 1) using a Rac1 interaction–impaired mutant RhoGDIa charged amino acids (Fig. 6A), inhibited Rac1 in a cell-free NADPH (D45/185A) (Fig. 4F), 2) using HEK293Nox2/FcgRIIa cells with Rac1 oxidase assay system using the membrane fraction of neutrophils as knockdown (Fig. 4G–I), 3) using a phagosome-targeting impaired oxidase assembly (29). However, a peptide of Thr7-Ile14 from human mutant Rac1(6A) (Fig. 5C, Supplemental Fig. 4E), and 2) using RhoGDIa, which contains only one negatively charged amino acid, RhoGDIa(5A) with coexpression of Rac1 (Supplemental Fig. 4C). was less effective (29). These data are consistent with our previous Furthermore, based on our previous report of the accumulation of report showing the dependence of Rac accumulation on phagosomes PA-producing PLD2 on phagosomes (26), we demonstrated that both and ROS production on the number of positively charged amino RhoGDIa-mKO and GFP-Rac1 accumulate on the phagosome when acids in the PB motif of Rac (15). Thus, the inhibitory effects of the PLD2 but not inactive PLD2(H442D) was overexpressed, and that negative charges found in the flexible N-terminal of RhoGDIa and PLD2 does not bind to Rac1 or RhoGDIa. Aside from the Rac1-PA RhoGDIb are likely mediated by masking of the PB motif in the C (produced by PLD2) interaction on RhoGDIa–Rac1 translocation, terminus of Rac, as suggested in a recent review (30). another mechanism that uses PA may function. During hepatocyte We previously demonstrated that an interaction between the growth factor–mediated membrane ruffling in Madin–Darby canine PB motif of Rac and anionic phospholipids (particularly PA and kidney cells, diacylglycerol kinase (DGK)a promoted the translo- phosphatidylinositol 3,4,5-triphosphate) is a key determinant in cation of atypical protein kinase Cz/i, stably associated with the Rac accumulation on phagosomes (15). Afterward, a study described RhoGDIa–Rac1 complex, to the PM through PA production (31). The Journal of Immunology 2567 Downloaded from

FIGURE 6. The negatively charged N terminus of RhoGDIb suppresses the Rac-dependent phagosomal targeting of RhoGDIb and ROS production. (A) B D B

Comparison of the amino acid sequence of human RhoGDIa and RhoGDIb.( – ) GFP-Rac1 was cotransfected with RhoGDIb-mKO ( ), RhoGDIb http://www.jimmunol.org/ (D42A)-mKO (C), or DN22-RhoGDIb-mKO (D) in RAW264.7 cells. The transfected cells were stimulated with 2-mm BIgG. Only GFP-Rac1, but not RhoGDIb-mKO (B) or RhoGDIb(D42A)-mKO (C), accumulates on phagosomes. In sharp contrast, DN22-RhoGDIb-mKO accumulates with GFP-Rac1 on phagosomes (D). The arrows and arrowheads indicate phagosomes engulfing BIgG. (E) Representative immunoprecipitation data (n $ 5) for the interac- tion between Myc-Rac1 and RhoGDIb-GFP, its mutants, or RhoGDIa-GFP. Right panel shows quantification of immunoprecipitated protein bands using ImageJ. (F) HEK293Nox2/FcgRIIa cells were transfected with a mock vector, RhoGDIa-GFP, RhoGDIb-GFP, RhoGDIb(D42A)-GFP, RhoGDIb(D182A)- GFP, or DN22-RhoGDIb-GFP in combination with Phox proteins and Myc-Rac1. The cells were stimulated with BIgG, and ROS release was measured (n $ 5). RhoGDIb and DN22-RhoGDIb show moderate suppression and RhoGDIb(D42A) and RhoGDIb(D182A) show faint suppression. The comparable expression of proteins was confirmed by immunoblotting. by guest on September 24, 2021

Additionally, it was reported that DGKz, stably associated with parently less than that of DN25-RhoGDIa (Supplemental Fig. 3D). PAK1 and RhoGDIa-Rac1, promoted the release of RhoGDIa from The residence time of RhoGDIa in the PM was not increased by Rac1 through DGKz→PA→PAK1–mediated RhoGDIa phosphory- Rac1. These results are consistent with the observed lack of phago- lation at platelet-derived growth factor–induced membrane ruffling somal RhoGDIa accumulation under confocal fluorescence imag- in fibroblasts (32). These studies support an involvement of PA pro- ing, even with Rac1 coexpression (Fig. 1C). Thus, a phagosomal duced from DGK in regulation of RhoGDIa–Rac1 translocation to accumulation enhancing factor for Rac1, such as PLD2, or neu- membranes and dissociation on membranes. trophils, which shows 10-fold or more ROS production and In contrast to no detection of RhoGDIa-mKO on phagosomes stronger phagosomal accumulation of Rac than RAW264.7 cells without coexpression of Rac1 and PLD2 under our confocal fluo- (15), is probably required to detect accumulation of RhoGDIa on rescence imaging, DN25-RhoGDIa-mKO and RhoGDIa(8A)- phagosomes under confocal fluorescence imaging. mKO accumulated on phagosomes even without coexpression The RhoGDIb–Rac1 complex is expected to be more steadily of Rac1; nevertheless, DN25-RhoGDIa and RhoGDIa(8A) retained in the cytoplasm due to the larger number of negatively maintained their abilities to bind Rac1 and to inhibit ROS produc- charged amino acids in the N terminus of RhoGDIb compared with tion similar to RhoGDIa. Although we cannot exclude the possi- RhoGDIa. However, the inhibitory effects of RhoGDIb on Rac1 bility that D25-RhoGDIa and RhoGDIa(8A)aremorestablewith translocation to phagosomes and ROS production were weaker than Rac1 on phagosomes owing to an undefined mechanism, the dis- those of RhoGDIa. Our imaging studies using RAW264.7 cells crepancy may be at least partially explained by the electrostatic revealed no apparent accumulation of RhoGDIb-mKO on phago- repulsion between the negatively charged N terminus of RhoGDIa somes, whereas the N-terminal deletion mutant D22-RhoGDIb- and the negative charge of the phagosome, which is enhanced by mKO accumulated on phagosomes along with GFP-Rac1. A study anionic phospholipids produced during phagocytosis. DN25- using the fluorescence resonance energy transfer technique in RhoGDIa and RhoGDIa(8A) have much weaker electrostatic re- RAW264.7 cells showed that RhoGDIb accumulates on phago- pulsion potency than does RhoGDIa; as a result, these proteins with somes during the phagocytosis of Listeria monocytogenes or the similar ability to bind Rac1 may remain longer on phagosomes Escherichia coli (33). These data suggest that RhoGDIb also with Rac1 and may keep the inhibitory effect on ROS production accumulates on phagosomes as a RhoGDIb–Rac1 complex sim- (Fig. 7B, 7C). In support of this proposal, our single molecule im- ilar to RhoGDIa, but the accumulation of the RhoGDIb is much aging showed prolonged residence time in the PM of D25-RhoGDIa weaker than that of RhoGDIa. The weaker accumulation of compared with RhoGDIa (Supplemental Fig. 3D). Single molecule RhoGDIb on phagosomes is likely due to its weaker binding af- imaging also showed that the frequency of RhoGDIa recruitment to finity for Rac1 and its stronger electrostatic repulsion on phago- the PM was slightly increased by Rac1 coexpression but was ap- somes than RhoGDIa. 2568 NEGATIVELY CHARGED FLEXIBLE N TERMINI OF RhoGDIa/b Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. The evolutionally conserved two clusters of negatively charged amino acids in N termini of RhoGDIs and their functional mechanisms. (A) Two clusters of negatively charged amino acids in N termini of RhoGDIs are conserved in many species, including plants. Plants have their own three specific RhoGDIs (1–3). The second cluster has a larger number of negatively charged amino acids than does the first cluster. The second cluster in RhoGDIb possesses a larger number of negatively charged amino acids than that in RhoGDIa.(B) Schematic comparison between human RhoGDIa and RhoGDIb with regard to the mechanisms of cytoplasm retention and membrane targeting. RhoGDIs do not feature any clear domains responsible for membrane targeting. (C) Model of Rac activation during phagocytosis. The RhoGDI–Rac complex translocates to the phagosome mediated by the in- teraction between the PB motif in the C terminus of Rac and the anionic phospholipids produced on the phagosome. Then, RhoGDI dissociates from Rac on the phagosome through RhoGDI dissociation factors. Note electrostatic repulsion between the negatively charged amino acids in the N terminus of RhoGDI and anionic phospholipids produced on phagosome.

In summary, the translocation/accumulation of RhoGDIa/b-Rac1 Disclosures to phagosomes and the inhibitory effect of RhoGDIa/b on ROS The authors have no financial conflicts of interest. production in Fcg-mediated phagocytosis may be regulated by a balance of the following three factors: 1) the negatively charged N-terminal of RhoGDIa/b, 2) the binding affinity of RhoGDIa/b References 1. Garcia-Mata, R., E. Boulter, and K. Burridge. 2011. The “invisible hand”: for Rac1, and 3) anionic phospholipids produced on phagosomes regulation of RHO GTPases by RHOGDIs. Nat. Rev. Mol. Cell Biol. 12: 493– (Fig. 7B). After the translocation of the RhoGDIa/b–Rac1 com- 504. plex to phagosomes, Rac1 must be released for its activation on 2. Dovas, A., and J. R. Couchman. 2005. RhoGDI: multiple functions in the reg- ulation of Rho family GTPase activities. Biochem. J. 390: 1–9. phagosomes, which is likely induced by RhoGDI dissociation 3. Takahashi, K., T. Sasaki, A. Mammoto, K. Takaishi, T. Kameyama, S. Tsukita, factors (1, 2) (Fig. 7C). and Y. Takai. 1997. Direct interaction of the Rho GDP dissociation inhibitor with The Journal of Immunology 2569

ezrin/radixin/moesin initiates the activation of the Rho small . J. Biol. 20. Ueyama, T., M. R. Lennartz, Y. Noda, T. Kobayashi, Y. Shirai, K. Rikitake, Chem. 272: 23371–23375. T. Yamasaki, S. Hayashi, N. Sakai, H. Seguchi, et al. 2004. Superoxide pro- 4. Yamashita, T., and M. Tohyama. 2003. The p75 receptor acts as a displacement duction at phagosomal cup/phagosome through bI protein kinase C during factor that releases Rho from Rho-GDI. Nat. Neurosci. 6: 461–467. FcgR-mediated phagocytosis in microglia. J. Immunol. 173: 4582–4589. 5. DerMardirossian, C., A. Schnelzer, and G. M. Bokoch. 2004. Phosphorylation of 21. Ueyama, T., T. Tatsuno, T. Kawasaki, S. Tsujibe, Y. Shirai, H. Sumimoto, RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol. Cell 15: 117–127. T. L. Leto, and N. Saito. 2007. A regulated adaptor function of p40phox: distinct 6. DerMardirossian, C., G. Rocklin, J. Y. Seo, and G. M. Bokoch. 2006. Phos- p67phox membrane targeting by p40phox and by p47phox. Mol. Biol. Cell 18: 441– phorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol- 454. membrane cycling. Mol. Biol. Cell 17: 4760–4768. 22. Ueyama, T., J. Nakakita, T. Nakamura, T. Kobayashi, J. Son, M. Sakuma, 7. Ugolev, Y., Y. Berdichevsky, C. Weinbaum, and E. Pick. 2008. Dissociation of H. Sakaguchi, T. L. Leto, and N. Saito. 2011. Cooperation of p40phox with Rac1(GDP)·RhoGDI complexes by the cooperative action of anionic liposomes p47phox for Nox2-based NADPH oxidase activation during Fcg receptor containing phosphatidylinositol 3,4,5-trisphosphate, Rac guanine nucleotide (FcgR)-mediated phagocytosis: mechanism for acquisition of p40phox phosphati- exchange factor, and GTP. J. Biol. Chem. 283: 22257–22271. dylinositol 3-phosphate (PI(3)P) binding. J. Biol. Chem. 286: 40693–40705. 8. Leto, T. L., S. Morand, D. Hurt, and T. Ueyama. 2009. Targeting and regulation 23. Ueyama, T., M. Geiszt, and T. L. Leto. 2006. Involvement of Rac1 in activation of reactive oxygen species generation by Nox family NADPH oxidases. Anti- of multicomponent Nox1- and Nox3-based NADPH oxidases. Mol. Cell. Biol. oxid. Redox Signal. 11: 2607–2619. 26: 2160–2174. 9. Nauseef, W. M. 2008. Biological roles for the NOX family NADPH oxidases. J. 24. Karasawa, S., T. Araki, T. Nagai, H. Mizuno, and A. Miyawaki. 2004. Cyan- Biol. Chem. 283: 16961–16965. emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for 10. Sumimoto, H. 2008. Structure, regulation and evolution of Nox-family NADPH fluorescence resonance energy transfer. Biochem. J. 381: 307–312. oxidases that produce reactive oxygen species. FEBS J. 275: 3249–3277. 25. Colley, W. C., T. C. Sung, R. Roll, J. Jenco, S. M. Hammond, Y. Altshuller, 11. Brunet, N., A. Morin, and B. Olofsson. 2002. RhoGDI-3 regulates RhoG and D. Bar-Sagi, A. J. Morris, and M. A. Frohman. 1997. Phospholipase D2, a dis- targets this protein to the Golgi complex through its unique N-terminal domain. tinct phospholipase D isoform with novel regulatory properties that provokes Traffic 3: 342–357. cytoskeletal reorganization. Curr. Biol. 7: 191–201. 12. Keep, N. H., M. Barnes, I. Barsukov, R. Badii, L. Y. Lian, A. W. Segal, 26. Cheeseman, K. L., T. Ueyama, T. M. Michaud, K. Kashiwagi, D. Wang, P. C. Moody, and G. C. Roberts. 1997. A modulator of rho family G proteins, L. A. Flax, Y. Shirai, D. J. Loegering, N. Saito, and M. R. Lennartz. 2006. rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flex- Targeting of protein kinase C-ε during Fcg receptor-dependent phagocytosis Downloaded from ible N-terminal arm. Structure 5: 623–633. requires the εC1B domain and phospholipase C-g1. Mol. Biol. Cell 17: 13. Grizot, S., J. Faure´, F. Fieschi, P. V. Vignais, M. C. Dagher, and E. Pebay- 799–813. Peyroula. 2001. Crystal structure of the Rac1-RhoGDI complex involved in 27. Dransart, E., A. Morin, J. Cherfils, and B. Olofsson. 2005. Uncoupling of in- NADPH oxidase activation. Biochemistry 40: 10007–10013. hibitory and shuttling functions of Rho GDP dissociation inhibitors. J. Biol. 14. Scheffzek, K., I. Stephan, O. N. Jensen, D. Illenberger, and P. Gierschik. 2000. Chem. 280: 4674–4683. The Rac-RhoGDI complex and the structural basis for the regulation of Rho 28. Nomanbhoy, T. K., and R. Cerione. 1996. Characterization of the interaction proteins by RhoGDI. Nat. Struct. Biol. 7: 122–126. between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J. Biol. Chem.

15. Ueyama, T., M. Eto, K. Kami, T. Tatsuno, T. Kobayashi, Y. Shirai, 271: 10004–10009. http://www.jimmunol.org/ M. R. Lennartz, R. Takeya, H. Sumimoto, and N. Saito. 2005. Isoform-specific 29. Golovanov, A. P., T. H. Chuang, C. DerMardirossian, I. Barsukov, D. Hawkins, membrane targeting mechanism of Rac during FcgR-mediated phagocytosis: R. Badii, G. M. Bokoch, L. Y. Lian, and G. C. Roberts. 2001. Structure-activity positive charge-dependent and independent targeting mechanism of Rac to the relationships in flexible protein domains: regulation of rho GTPases by RhoGDI phagosome. J. Immunol. 175: 2381–2390. and D4 GDI. J. Mol. Biol. 305: 121–135. 16. Yeung, T., B. Heit, J.-F. Dubuisson, G. D. Fairn, B. Chiu, R. Inman, A. Kapus, 30. Dransart, E., B. Olofsson, and J. Cherfils. 2005. RhoGDIs revisited: novel roles M. Swanson, and S. Grinstein. 2009. Contribution of phosphatidylserine to in Rho regulation. Traffic 6: 957–966. membrane surface charge and protein targeting during phagosome maturation. J. 31. Chianale, F., E. Rainero, C. Cianflone, V. Bettio, A. Pighini, P. E. Porporato, Cell Biol. 185: 917–928. N. Filigheddu, G. Serini, F. Sinigaglia, G. Baldanzi, and A. Graziani. 2010. 17. Garin, J., R. Diez, S. Kieffer, J. F. Dermine, S. Duclos, E. Gagnon, R. Sadoul, Diacylglycerol kinase a mediates HGF-induced Rac activation and membrane C. Rondeau, and M. Desjardins. 2001. The phagosome proteome: insight into ruffling by regulating atypical PKC and RhoGDI. Proc. Natl. Acad. Sci. USA phagosome functions. J. Cell Biol. 152: 165–180. 107: 4182–4187.

18. Robinson, J. M., and J. A. Badwey. 2002. Rapid association of cytoskeletal 32. Abramovici, H., P. Mojtabaie, R. J. Parks, X. P. Zhong, G. A. Koretzky, by guest on September 24, 2021 remodeling proteins with the developing phagosomes of human neutrophils. M. K. Topham, and S. H. Gee. 2009. Diacylglycerol kinase z regulates actin Histochem. Cell Biol. 118: 117–125. reorganization through dissociation of Rac1 from RhoGDI. Mol. 19. Dovas, A., Y. Choi, A. Yoneda, H. A. Multhaupt, S. H. Kwon, D. Kang, E. S. Oh, Biol. Cell 20: 2049–2059. and J. R. Couchman. 2010. Serine 34 phosphorylation of rho guanine dissoci- 33. Wu, W., Y. M. Hsu, L. Bi, Z. Songyang, and X. Lin. 2009. CARD9 facilitates ation inhibitor (RhoGDIa) links signaling from conventional protein kinase C to microbe-elicited production of reactive oxygen species by regulating the LyGDI- RhoGTPase in cell adhesion. J. Biol. Chem. 285: 23296–23308. Rac1 complex. Nat. Immunol. 10: 1208–1214.