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Platelet-Activating Factor-Induced Clathrin-Mediated Endocytosis Requires β -Arrestin-1 Recruitment and Activation of the p38 MAPK Signalosome at the Plasma This information is current as Membrane for Actin Bundle Formation of September 24, 2021. Nathan J. D. McLaughlin, Anirban Banerjee, Marguerite R. Kelher, Fabia Gamboni-Robertson, Christine Hamiel, Forest R. Sheppard, Ernest E. Moore and Christopher C. Silliman

J Immunol 2006; 176:7039-7050; ; Downloaded from doi: 10.4049/jimmunol.176.11.7039 http://www.jimmunol.org/content/176/11/7039 http://www.jimmunol.org/ References This article cites 49 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/176/11/7039.full#ref-list-1

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Platelet-Activating Factor-Induced Clathrin-Mediated Endocytosis Requires ␤-Arrestin-1 Recruitment and Activation of the p38 MAPK Signalosome at the Plasma Membrane for Actin Bundle Formation1

Nathan J. D. McLaughlin,* Anirban Banerjee,† Marguerite R. Kelher,† Fabia Gamboni-Robertson,† Christine Hamiel,† Forest R. Sheppard,†‡ Ernest E. Moore,†‡ and Christopher C. Silliman2*†§

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals.

We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of ␤-arres- Downloaded from tin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 ␮M PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, ␤-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with ␤-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, ␤-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK http://www.jimmunol.org/ kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of ␤-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a ␤-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling

requires CME, ␤-arrestin-1 recruitment of a p38 MAPK signalosome, and speciﬁc actin bundle formation at the PAFR for by guest on September 24, 2021 transduction before endosomal scission. The Journal of Immunology, 2006, 176: 7039–7050.

he G protein-coupled receptors are ubiquitous, and bind- subunits (␣ and ␤␥) (5). These subunits cause the release of cal- ing of the ligand may cause receptor internalization via cium stores and the activation of small GTPases and MAPK sig- T clathrin-mediated endocytosis (CME),3 which activates naling cascades (5). Within seconds, these signals can be termi- speciﬁc signaling pathways (1). Classically, CME consists of three nated by members of the arrestin family by their binding to the steps: receptor desensitization, sequestration of receptors to clath- phosphorylated C terminus (cytosolic) region of the receptor and rin-coated pits, and receptor internalization (2, 3). However, there uncoupling it from the associated heteromeric G proteins (2). In is increasing evidence for discrete signal transduction at the site of addition, arrestins may have a role in receptor internalization and the receptor for each step in CME (2, 4). The initial signal is nonreceptor tyrosine kinase activation (4, 6). transduced via coupling of the receptor to heteromeric G protein Cell motility is essential for host defense, especially for leukocyte recruitment to the site of injury. Polymorphonuclear neutro-

*Department of Pediatrics and †Department of Surgery, University of Colorado phils (PMNs) play a key role in host defense, and exert their mi- School of Medicine, Denver, CO 80262; ‡Department of Surgery, Denver Health crobicidal function in the tissues. The orderly process of migration Medical Center, Denver, CO 80204; and §Bonﬁls Blood Center, Denver, CO 80230 from the vasculature to the tissue initiates PMN priming, which Received for publication August 16, 2005. Accepted for publication March 13, 2006. can be facilitated by a number of proinﬂammatory protein and

The costs of publication of this article were defrayed in part by the payment of page lipid mediators, including IL-8, leukotriene B4, and platelet-acti- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vating factor (PAF) (7–10). PMN priming is characterized by 1 distinct physiological changes, including increased actin polymer- This work was supported by Bonfils Blood Center, Grant HL-59355-06 from the ␤ National Heart, Lung, and Blood Institute and Grant GM-49222 from the National ization resulting in shape change, firm 2 integrin-mediated ad- Institute of General Medical Sciences. herence to the vascular endothelium, and augmentation of the mi- 2 Address correspondence and reprint requests to Dr. Christopher C. Silliman, crobicidal response, both oxidative and nonoxidative, to a Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230. E-mail address: [email protected] subsequent stimulus. In addition, PMN priming agents are etio- logic in two event models of PMN-mediated acute lung injury, 3 Abbreviations used in this paper: CME, clathrin-mediated endocytosis; PMN, polymorphonuclear neutrophil; PAF, platelet-activating factor; PAFR, PAF receptor; sepsis, and multiple organ failure (11, 12). ASK1, apoptosis signal-regulating kinase-1; MKK, MAPK kinase-3; MAPKAPK-2, Ligation of the PAF receptor (PAFR) recruits members of the MAPK-activated protein kinase-2; LSP-1, leukocyte-specific protein-1; FRET, fluo- ␤ rescent resonance energy transfer; PAK, p21-activated kinase; ARP, actin-related arrestin family ( -arrestin-1), which desensitizes the receptor, protein; TAK, TGF-␤ activated-kinase-1. operationally defined as the inability of repeated exposures to a

stimulus to cause multiple signaling events, e.g., the release of strument filter control. Images were acquired using Intelligent Imaging stored Ca2ϩ (13). However, the role of these arresting proteins to Innovations SlideBook software. All images compared within a single fig- ␮ facilitate signaling remains undefined. We hypothesize that PAF ure were acquired as Z-stacks in 0.2- m intervals, and the planes are as described in the figures. All Z-stack images were deconvolved by applying causes CME of its receptor and before endosomal scission elicits constrained iterative deconvolution and Gaussian noise smoothing from ␤-arrestin-1-mediated recruitment and activation of the p38 system specific point spread functions (17). Following deconvolution, im- MAPK signalosome and discrete actin reorganization at the PAFR, ages were cropped to represent the middle most planes (center Ϯ 10 all required for CME and distinct from actin that participates in planes) and the proteins in question masked to represent zero fluorescence in IgG negative controls. Cell polarization was assessed using Nomarski cell polarization and chemotaxis. differential interference contrast microscopy (Nomarski images), and for these images, microscopy planes were taken through the pseudopodia as Materials and Methods described in the figures (18). Materials Fluorescent resonance energy transfer (FRET) microscopy Unless otherwise indicated, all chemicals were purchased from Sigma- Aldrich. All reagents were endotoxin-free, and all solutions were made FRET determinations were obtained by direct acceptor photobleaching from sterile water (Pharmacopeia). Acrylamide, N,NЈ-methylene-bis-acryl- FRET as previously described (19). Within this context, the ability of the amide, and TEMED were obtained from Bio-Rad. ECL reagents were ob- two secondary Abs to FRET were acquired between Rhodamine (excitation tained from Amersham. Nitrocellulose paper, x-ray film, and Nunc 96-well wavelength, 550 nm; emission, 570 nm, acquired on the Cy3 channel; plates were purchased from Life Science Products, and indo-1-AM from Jackson ImmunoResearch Laboratories) and Alexa Fluor 488 (excitation, Molecular Probes. Abs to the clathrin H chain (H-300), PAFR (H-300, 495 nm; emission, 519 nm, acquired on the FITC channel (Molecular N-20), ␤-arrestin-1 (K-16), dynamin-2 (H-300), TGF-␤ activated-kinase Probes), except in the case of FRETs between clathrin H chain and the PAFR, which were determined using Alexa Fluor 647 (excitation, 650 nm; (TAK)-1 (M-579), apoptosis signal-regulating kinase-1 (ASK1, F-9, Downloaded from H-300), phospho-MAPK kinase-3 (MKK3)/6 (B-9), leukocyte-specific emission, 668 nm, acquired on the Cy5 channel; Molecular Probes) and protein-1 (LSP-1, N-20), secondary anti-goat IgG-HRP, and anti-mouse Alexa Fluor 488. In all cases, an initial image was acquired of the donor IgG-HRP, and protein A/G Plus Sepharose beads were purchased from and acceptor channels, and following capture a region of interest was de- Santa Cruz Biotechnology. Dual-phosphorylated p38 MAPK (Thr180/ fined, a mask applied, and the specified acceptor (Cy5 or Cy3) ablated (i.e., Tyr182), p21-activated kinase (PAK)1, PAK3, and secondary anti-rabbit photobleaching, per manufacturer’s nomenclature). Ablation was accom- IgG-HRP were obtained from Cell Signaling Technology, and phospho- plished using a Photonics FRAP laser fitted with the appropriate wave- serine and pan-arrestin Abs were purchased from Zymed Laboratories and length discriminator (Rhodamine 610 (Cy5) or Rhodamine 540 (Cy3)). FRET efficiencies (E ) were calculated using the following formula: E ϭ http://www.jimmunol.org/ Abcam, respectively. Anti-PAFR for immunohistochemistry was obtained i i [(I Ϫ I )/(I )], where I is the mean intensity of the donor pre- from Cayman Chemicals. Anti-actin-related protein (Arp)3 was obtained post,i pre,i pre,i pre,i photobleach image and I is the mean intensity of the donor postpho- from Upstate Biotechnology and phospho-LSP-1 (Ser204) from MBL post,i International. tobleach image (19). Images are displayed in pseudocolor in which blue is cold (little FRET) and red is hot (most FRET). PMN isolation Intracellular neutralization of specific proteins Heparinized whole blood was drawn from healthy human volunteers by BioPorter was reconstituted according to the manufacturer’s directions. venipuncture consent using a protocol approved by the Combined Internal Briefly, individual tubes were reconstituted to a total volume of 40 ␮l with Review Board and Human Subject Committee at the University of Colo- KRPD Ϯ 4 ␮g of the Ab specified in the text for 5 min at room temper- rado School of Medicine, and PMNs were isolated as previously described 6 ature. PMNs (5 ϫ 10 cells) were incubated with buffer, vehicle only, or by guest on September 24, 2021 (14). Cells were resuspended to a concentration of 2.5 ϫ 107 cells/ml in vehicle with the Ab for2hat37°C. Following incubation, PMNs were Krebs-Ringers phosphate buffer (pH 7.35) with 2% dextrose (KRPD) and centrifuged for 3 min at 1800 rpm at 4°C, and resuspended to 5 ϫ 106 used immediately for all subsequent manipulations. cells/ml. To control for IgG introduction, a FITC-IgG control was intro- Whole cell lysate, immunoprecipitation, and discontinuous duced, and Z-stack images acquired as described. ␤-arrestin-1 and dy- subcellular fractionation namin-2 are sufficient for native proteins as demonstrated by use in immunoprecipitation. Furthermore, for intracellular neutralization two PMNs (1.25 ϫ 107 cells (whole cell lysate and immunoprecipitation) or different Abs to different epitopes were used. 1 ϫ 108 cells (subcellular fractionation)) were incubated with buffer or 2 ␮M PAF for 1 min. Reactions were stopped with the addition of ice-cold Statistical analysis relaxation buffer (10 mM PIPES (pH 7.4), 3 mM NaCl, 100 mM KCl, 3.5 Statistical analysis was performed by paired ANOVA with Bonferroni/Dun ␮ ␮ MgCl2, 1.2 mM EGTA, 10 g/ml leupeptin, and 50 g/ml PMSF) and post hoc analysis using p Ͻ 0.05 or p Ͻ 0.01 as the level of statistical ϫ immediately sonicated (2 30 s). Lysates were cleared and used for West- significance. ern blot analysis, immunoprecipitation, or subjected to subcellular fractionation as previously described (15). Purity of fractions was determined Results using lactate dehydrogenase as a cytosolic marker, L-selectin as a membrane marker, and myeloperoxidase as a granular marker. Proteins were Proximal interaction of PAFR with CME proteins separated by SDS-PAGE (7.5 or 10%) acrylamide gel with SDS electro- To investigate whether PAF-mediated PAFR ligation causes CME, phoresis, transferred to a nitrocellulose membrane, and these membranes were cross-linked and probed with the specific Abs indicated. we established a relationship between the PAFR and known CME proteins. Immunoprecipitation of ␤-arrestin-1 coprecipitated both Cytosolic calcium measurements clathrin and the monomeric (40 kDa) and dimeric (80 kDa) con- Cytosolic Ca2ϩ concentrations were measured by indo-1-AM loading of formations of the PAFR (Fig. 1A and data not shown) (20). Fur- PMNs and analysis in a PerkinElmer LS50B spectrofluorimeter over real- thermore, ␤-arrestin-1 coprecipitated the activated, serine phos- time, as previously described (16). Briefly, PMNs (2 ϫ 106) were loaded phorylated PAFR (Fig. 1B). The interaction between the PAFR ␮ with 5 M indo-1-AM for 7 min in the dark, centrifuged, and resuspended and ␤-arrestin-1 were further defined by acceptor photobleaching in warm KRPD. PMNs were placed in a PerkinElmer LS50B with constant ␤ stirring, and the calcium concentrations were measured in real-time with FRET (Fig. 1C). The interaction between -arrestin-1 (Fig. 1C, excitation at 355 nm and dual emission wavelengths were monitored at 410 vii–ix; red) and the PAFR (Fig. 1C, x–xii; green) was FRET pos- and 470 nm. Data were processed using Grynkiewicz equation. itive following PAF stimulation (1 min) as compared with resting Digital fluorescent microscopy cells. Moreover, there was a PAF-induced FRET positive interaction between the clathrin H chain (Fig. 1D, i–iii; blue) and both 5 PMNs (5.0 ϫ 10 cells) were warmed to 37°C and then incubated with ␤-arrestin-1 (Fig. 1D, iv–vi; red) and the PAFR (Fig. 1D, vii–ix; either buffer or 2 ␮M PAF for 1 min and prepared as previously described (16). Images were acquired with a Zeiss Axiovert fitted with a Cooke CCD green). It was possible to use clathrin H chain as the final acceptor SensiCam camera using a Chroma Technology Sedat with single excitation based on the overlap of its spectra with Cy3 or FITC. Furthermore, and emission filters and a multiple bandpass dichroic filter and Sutter In- immunoprecipitation of dynamin-2, a well-described mediator in The Journal of Immunology 7041 Downloaded from http://www.jimmunol.org/

FIGURE 1. PAF ligation recruits components of CME. A, PMNs were stimulated with buffer or 2 ␮M PAF for 1 min, and immunoprecipitation of whole cell lysates was performed using Abs to ␤-arrestin-1 (buffer and PAF) or isotypic IgG controls (goat; Iso). Western blots probed for the PAFR (40 and 80 kDa), clathrin H chain (200 kDa), and ␤-arrestin-1 (50 kDa). PAF-induced coprecipitation of the PAFR and clathrin H chain following PAF treatment, as compared with buffer-treated PMNs (n ϭ 5). B, Probing ␤-arrestin-1 immunoprecipitates for phosphoserine demonstrated similar bands as in A at 40 and 80 kDa (n ϭ 5). C, The interaction between ␤-arrestin-1 (acceptor; i–iii, vii–ix; red) and the PAFR (donor; iv–vi, x–xii; green) was analyzed using acceptor photobleaching FRET. An initial image was acquired of both the acceptor channel (Cy3; i and vii) and donor channel (FITC; iv and x) followed by Cy3 photobleaching using Coumarin 540 nm dye cube within only the region of interest (deﬁned by the white square) being photobleached (ii, v, viii, and xi).

A second image was acquired at the same x, y, z coordinates and the effect of photobleaching is demonstrated by subtraction of the second image (Post) by guest on September 24, 2021 from the first (Pre) (iii and ix), which found no photobleaching outside of the defined region of interest. FRET-positive images were determined by the formula described in Materials and Methods, demonstrating that following PAF treatment (ϩPAF) there was a positive FRET interaction (xii) between ␤-arrestin-1 and the PAFR as compared with resting cells (vi) (scale bar, 7 ␮m; Ͼ20 cells in two independent experiments). D, FRET analysis was done using techniques similar to those used in C, but the clathrin H chain (acceptor; i–iii; blue) was photobleached using a Rhodamine 610 dye cube. Photobleaching Cy5 demonstrated a FRET positive interaction between clathrin H chain and both ␤-arrestin-1 (donor; iv–vi; red) and the PAFR (donor; vii–ix; green) (scale bar, 7 ␮m; Ͼ20 cells in two independent experiments). E, Immunoprecipitation (IP) of whole cell lysates using Abs to dynamin-2 (buffer and PAF) or IgG-isotype controls (goat; Iso) demonstrated coprecipitation of the dimer PAFR (80 kDa), as compared with buffer-treated PMNs (n ϭ 3). F, PMNs were stimulated with buffer or 2 ␮M PAF for 1 min, subcellular fractions prepared, and the cytosol and membrane fractions immunoprecipitated using Abs to ␤-arrestin-1 (buffer and PAF). Western blots probed for the PAFR and ␣-adaptin. PAF-induced coprecipitation of the PAFR and ␣-adaptin in the membrane fraction following PAF treatment is shown, as compared with buffer-treated PMNs and the cytosol from both resting PMNs and PAF-treated PMNs (n ϭ 5). G, Subcellular fractions were prepared from resting PMNs, and Western blots probed for proteins specific for the cytosol (lactate dehydrogenase), membrane (L-selectin), and granules myeloperoxidase (MPO) (n ϭ 3).

CME, in PAF-stimulated PMNs also elicited the coprecipitation of 2, neutralizing Abs against ␤-arrestin-1 or an IgG isotype control the dimeric PAFR (Fig. 1E). In addition, immunoprecipitation of were intracellularly introduced using BioPorter, a liposomal-based ␤-arrestin-1 from membrane fractions demonstrated PAF-depen- delivery system (21). Intracellular neutralization of ␤-arrestin-1 dent coprecipitation of the AP-2 protein, ␣-adaptin, a marker of resulted in a loss of desensitization, as evidenced by undiminished CME, and the PAFR (Fig. 1F). Additionally, to demonstrate our Ca2ϩ mobilization in response to repeated PAF treatments. In con- subcellular preparation proteins known to be found in the cytosol, trast, intracellular introduction of an IgG isotype control had no membrane and granules were probed for in subcellular fractions effect on receptor desensitization with PAF stimulation. prepared from resting PMNs (Fig. 1G). To determine whether PAFR internalization was ␤-arrestin-1 and dynamin-2 dependent, neutralizing Abs to ␤-arrestin-1, dy- 2ϩ Characterization of CME-dependent PAFR Ca mobilization namin-2, or an IgG isotype control were introduced into PMNs, and receptor internalization stimulated with buffer or PAF, and then smeared onto slides but Because Ca2ϩ mobilization is a G protein-coupled receptor-medi- the cells were not permeabilized (Fig. 3A). Using an Ab to an ated event and repeated stimulations by the same agent causes the extracellular epitope on the N terminus of the PAFR, we assessed receptor to become desensitized, i.e., refractory to repeated expo- the amount of peripheral membrane-associated receptor. Com- sures to the same agonist, we investigated the role of ␤-arrestin-1 pared with resting IgG isotype cells (Fig. 3A, i), there was a in receptor desensitization and PAFR internalization (13). In Fig. decrease in membrane-associated PAFR in the PAF-treated 7042 PAF CME REQUIRES ␤-ARRESTIN IN p38-INDUCED ACTIN BUNDLES

restin-1 or dynamin-2 (22). Whole cell lysates from PAF stimulated (1 min) were immunoprecipitated with an Ab against ␤-arrestin-1, which coprecipitated both p38 MAPK and the upstream regulator MKK3, and was not present in controls or immunoprecipitation with an IgG isotype (Fig. 4A). PAF stimulation of PMNs also demonstrated colocalization (Fig. 4B, viii, shown in pseudocolor) of dual phosphorylation (Thr180/Tyr182) of p38 MAPK, phospho-p38 MAPK (Fig. 4B, i and vi; green) with ␤-arrestin-1 (Fig. 4B, ii and vii; red) at the periphery of the cell, which was not present in resting cells (Fig. 4B, iii). Similarly, phosphorylated MKK3 (Fig. 4C, i and vi; green) translocated to the plasma membrane and had some colocalization with ␤-arrestin-1 (Fig. 4C,ii and vii; red) following PAF treatment (Fig. 4C, viii, shown in pseudocolor). Furthermore, the distribution of total p38 MAPK (Fig. 4B, iv and ix) was similar to phospho-p38 MAPK following FIGURE 2. ␤ -arrestin-1-dependent desensitization of the PAFR. Cells ␤ were incubated with BioPorter loaded with a neutralizing Ab to ␤-arres- treatment with PAF, which colocalized with -arrestin-1 at the tin-1 (␤-ar) Ab (solid line) or isotypic (Iso) control IgG (dashed line) for periphery of the cell (Fig. 4B, x, shown in pseudocolor). Total 2 h, and subsequently loaded with the Ca2ϩ indicator indo-1-AM. Stored MKK3 (Fig. 4C, iv and ix) demonstrated similar results, colocal- ϩ Ca2 release was monitored for 5 min, with PAF stimulations at 30 s (1) izing with ␤-arrestin-1 following PAF treatment (Fig. 4C,x, Downloaded from and 180 s (2) indicated by arrows. Cells incubated with IgG (dashed line) shown in pseudocolor). This colocalization was further conﬁrmed demonstrated normal desensitization indicated by the presence of a single with FRET analysis, which demonstrated a FRET-positive, phys- 2ϩ increase in cytosolic Ca release (PAF for 30 s). In contrast, Abs against ical interaction between phospho-MKK3 (Fig. 4D, ii and iii) and ␤-arrestin-1 (solid line) inhibited PAFR desensitization as demonstrated by ␤ 2ϩ -arrestin-1 (Fig. 4D, v and vi) following PAF treatment (Fig. 4D, a second increase in cytosolic Ca release following a second PAF stim- vii). Furthermore, immunoprecipitation of ␤-arrestin-1 from sub- ulation (PAF for 180 s) (n ϭ 4).

cellular fractions of PAF-treated PMNs demonstrated (phospho- http://www.jimmunol.org/ p38 MAPK) in the membrane fraction (Fig. 4E), conﬁrming the IgG-loaded cells (Fig. 3A, ii), whereas intracellular neutralization microscopy data that suggested that phosphorylation of p38 of ␤-arrestin-1 (Fig. 3A, iii) and dynamin-2 (Fig. 3A, iv) had mem- MAPK at 1 min occurred primarily in the membrane not within the brane associated PAFR levels identical with the resting cells, and cytosol. In contrast to previous studies, PAF did not cause p38 these differences were statistically signiﬁcant as compared with the MAPK to coprecipitate with dynamin-2 (Fig. 4F). PAF-treated IgG-loaded controls (Fig. 3B). Figures are represen- ␤ tative of cross-sections of a middle plane of PMNs. -arrestin-1-dependent but dynamin-2-independent p38 MAPK activation by guest on September 24, 2021 Recruitment of the p38 MAPK signaling cassette Because of the observed physical interaction between the p38 ␤-arrestin-1 and dynamin-2 can recruit and activate members of MAPK signalosome and ␤-arrestin-1 and because dynamin-2 may the MAPK family (2). Because PAF causes rapid activation of p38 be important in the activation of MAPKs, we investigated the role MAPK, we sought to determine its possible association with ␤-ar- of these mediators of CME on p38 MAPK activation (23, 24).

FIGURE 3. ␤-arrestin-1-and dynamin-2-dependent PAFR endocytosis. A, Cells were incubated with BioPorter loaded with a neutralizing Ab to ␤-arrestin-1 (iii), dynamin-2 (iv), or an IgG isotype control (i and ii) for 2 h, and subsequently stimulated with buffer (i) or 2 ␮M PAF (ii–iv). Slides of nonpermeabilized cells labeled with a N terminus-speciﬁc Ab to the PAFR were visualized, and demonstrated internalization of the PAFR following PAF treatment of PMNs (ii), whereas introduction of a ␤-arrestin-1 Ab (iii) or a dynamin-2 Ab (iv) demonstrated expression levels similar to buffer-treated IgG controls (i) (scale bar, 7 ␮m; Ͼ20 cells in two independent experiments). B, Statistical analysis of PAFR immunoreactivity in the membranes (voxel occupation), measured by the percentage of receptor surface expression, demonstrated signiﬁcant differences between the IgG PAF-treated cells and the IgG buffer-treated controls, ␤-arrestin-1 Ab cells, and dynamin-2 Ab cells. Results represent the mean Ϯ SEM from Ͼ20 cells in two independent .p Ͻ 0.05, using ANOVA ,ء .experiments The Journal of Immunology 7043 Downloaded from http://www.jimmunol.org/

FIGURE 4. ␤-arrestin-1 recruits MKK3/p38 MAPK with activation at the site of recruitment. A, Immunoprecipitation of ␤-arrestin-1 from whole cell lysates demonstrated coprecipitation of both p38 MAPK (38 kDa) and MKK3 (35 kDa) following PAF treatment, which was not present in buffer-treated PMNs or in those subjected to immunoprecipitation with IgG (n ϭ 4). B, Digital microscopy of dual phosphorylation (Ser/Thr) of p38 MAPK (p-p38 by guest on September 24, 2021 MAPK) (i and vi) and ␤-arrestin-1 (ii and vii) in ﬁxed cells showed very little phospho-p38 MAPK in resting cells (i) with equal distribution of ␤-arrestin-1 (ii), resulting in negligible colocalization (iii). Following 1 min of PAF treatment, phospho-p38 MAPK immunoreactivity was increased (iv), ␤-arrestin-1 was restricted to the periphery (vii), and there was colocalization shown in pseudocolor (viii). B, The distribution of total p38 MAPK (iv and ix) was similar to phospho-p38 MAPK following treatment with PAF, which colocalized with ␤-arrestin-1 at the periphery of the cell (x, shown in pseudocolor) (scale bar, 7 ␮m). Data are representative of three independent experiments. C, Similar spatial distribution was visualized in ﬁxed PMNs with very little phospho- MKK3 in resting cells as compared with PAF treated cells (i and vi). There is translocation of ␤-arrestin-1 to the cell periphery with PAF treatment (ii and vii), resulting in modest colocalization between ␤-arrestin-1 and phospho-MKK3 shown in pseudocolor (viii). Total MKK3 (iv and ix) demonstrated similar results, colocalizing with ␤-arrestin-1 following PAF treatment (x, shown in pseudocolor) (scale bar, 7 ␮m). Data are representative of three independent experiments. D, Acceptor photobleaching FRET was performed between ␤-arrestin-1 (acceptor; red) and phospho-MKK3 (donor; green), in identical fashion as that described in Fig. 1C. Resting cells (i) demonstrated very little phospho-MKK3, therefore FRET was not performed on buffer-treated controls. PAF-treated cells (ii–vii), however, found a positive FRET interaction between ␤-arrestin-1 and phospho-MKK3 (vii) (scale bar, 7 ␮m; Ͼ20 cells in two independent experiments). E, Discontinuous sucrose fraction were used to prepare pure cellular cytosol and membrane fractions, which were immunoprecipitated using an Ab against ␤-arrestin-1. Probing Western blots for phospho-p38 MAPK demonstrated immunoreactivity occurring only in the membrane fraction following PAF treatment as compared with resting membrane fractions and both resting and PAF-treated cytosols (n ϭ 4). Normal S.C., normal subcellular. F, Immunoprecipitation of whole cell lysates using an Ab against dynamin-2 exhibited no coprecipitation of p38 MAPK from either buffer or PAF treatment, but demonstrated immunoreactivity in the postimmunoprecipitation supernatant (n ϭ 3).

Using phospho-p38 MAPK as a readout, neutralizing Abs against immunoprecipitated from whole cell lysates (Fig. 6A). The immu- ␤-arrestin-1 and dynamin-2 or isotype IgG controls were intro- noprecipitates were probed with speciﬁc Abs to known MAP3K duced, and whole cell lysates were probed for phospho-p38 MAPK activators of MKK3: p21 (Cdc42/Rac)-activated kinase 1/3 and p38 MAPK (Fig. 5). Intracellular introduction of Abs speciﬁc (PAK1/3), TAK1, and ASK1. In contrast with previous reports, to ␤-arrestin-1 abrogated PAF-mediated p38 MAPK activation, PAF stimulation did not cause PAK1/3 (25) or TAK coprecipita- whereas introduction of IgG isotype controls did not affect PAF- tion with MKK3 (26) (data not shown, available upon request). mediated activation of p38 MAPK (Fig. 5). Furthermore, intracel- Rather, PAF-elicited ASK1 coprecipitation with MKK3, which lular neutralization of dynamin-2 did not inhibit p38 MAPK acti- was absent in buffer-treated controls (Fig. 6A). Surprisingly, there vation and demonstrated a similar amount of phospho-p38 MAPK was constitutive binding between p38 MAPK and ASK1, which compared with the isotype control (Fig. 5). was unchanged with PAF treatment (Fig. 6B). Because ASK1 is reported to primarily reside in the cytosol, subcellular fractions ASK1 is the proximal kinase for p38 MAPK activation were prepared and ASK1 was immunoprecipitated from the cy- To determine the MAPK kinase kinase (MAP3K) upstream of tosol and membrane (15). PAF caused coprecipitation of both MKK3, PMNs were treated with PAF (1 min) and MKK3 was phospho-MKK3 and phospho-p38 MAPK with ASK1 in both the 7044 PAF CME REQUIRES ␤-ARRESTIN IN p38-INDUCED ACTIN BUNDLES

FIGURE 5. ␤-arrestin-1-dependent but dynamin-2-independent p38 MAPK activation. PMNs were incubated with buffer or BioPorter loaded with 4 ␮g of isotype IgG (Iso), dynamin-2 Ab (Dyn), or ␤-arrestin-1 Ab (␤-ar), and subsequently stimulated with buffer (Resting) or 2 ␮M PAF. Introduction of the Abs did not cause p38 MAPK phosphorylation (p-p38 MAPK), but PAF treatment caused an increase in the phosphorylation of p38 MAPK in the IgG isotype-loaded control, which was abrogated by the intracellular introduction of the ␤-arrestin-1 Ab and unaffected by introduction of the dynamin-2 Ab (n ϭ 3).

FIGURE 6. Assembly and activation of a p38 MAPK signaling cassette. plasma membrane and the cytosol, and in control PMNs these PMNs were stimulated with buffer or 2 ␮M PAF for 1 min. Whole cell Downloaded from interactions were absent in the membrane and comparatively di- lysates immunoprecipitated for MKK3 (A) found coprecipitation of ASK1 minished in the cytosol (Fig. 6C). following PAF treatment, which was not present in buffer-treated controls (n ϭ 3). B, Immunoprecipitation of p38 MAPK found coprecipitation of PAF-mediated actin rearrangement ASK1 in both PAF-treated and buffer-treated cells (n ϭ 3). C, The immuno- Many chemoattractants cause rapid actin rearrangement, and the precipitation of ASK1 from subcellular fractions (cytosol and membrane) from PMNs stimulated with buffer or PAF and probed for phospho-p38 MAPK and effect of PAF on F-actin rearrangement was visualized by digital http://www.jimmunol.org/ microscopy (Fig. 7A). Consistent with previous studies, compari- phospho-MKK3 found coprecipitation of both phospho-p38 MAPK and phospho-MKK3 in both the cytosol and membrane fractions following son of the Nomarski images (18) (Fig. 7A, iii) with fluorescent PAF treatment (n ϭ 3). images (Fig. 7A, iv and v) demonstrated actin polymerization at the pseudopod following PAF treatment (Fig. 7A, white arrows). In addition, when the angle between two actin filaments with the same point of origin was measured there was an ϳ70° angle, char- tion of ␤-arrestin-1 significantly inhibited actin bundle formation acteristic of ARP2/3-mediated actin polymerization (18) (Fig. 7A, as well as cell polarization (Fig. 8A, ix-xii), similar to the IgG vi). Present also were punctate actin formations on the periphery of isotype in resting cells (Fig. 8A, i-iv). Furthermore, abrogation the cell body distinct from the PAF-induced F-actin in the pseu- with ␤-arrestin-1 Abs was statistically different from PAF-treated by guest on September 24, 2021 dopodia (Fig. 7A, v). These PAF-mediated peripheral F-actin ac- IgG isotype cells (Fig. 8B, i and ii). Neutralization of dynamin-2 cumulations in the cell body consisted of actin bundles because had no effect on actin bundle formation, similar to the IgG isotype they displayed distinct immunoreactivity with an Ab specific for (data not shown). an epitope present only during actin bundle formation (Fig. 7B, iv), To investigate whether the p38 MAPK signaling cassette was which were not present in a cross-section of the pseudopodia (Fig. necessary for actin bundle formation at the receptor membrane 7B, v). To better define cell polarization and active actin polymer- interface, PMNs were incubated with the specific p38 MAPK in- ization, slides were prepared looking at ARP3, a component of the hibitor SB203580 (1 ␮M) for 5 min before PAF or buffer stimu- actin polymerization protein ARP2/3 (Fig. 7C). Following PAF lation. Inhibition of p38 MAPK demonstrated a marked decrease treatment (Fig. 7C, iii and iv) there was a dramatic shift in ARP3 in actin bundle formation (Fig. 8C, xi) as well as cell polarization from the cytosol and periphery (Fig. 7C, ii) to the pseudopod (Fig. (Fig. 8C, ix) as compared with DMSO-pretreated, PAF-stimulated 7C, iii and iv). cells (Fig. 8C, v–viii). These results are similar to those seen with the intracellular neutralization of ␤-arrestin-1, for this neutraliza- ␤ -arrestin-1-dependent but dynamin-2-independent actin tion inhibited both actin bundle formation and polarization, differ- rearrangement ent from PAF-treated cells (Fig. 8D, i and ii). Because p38 MAPK To determine the importance of CME scaffold protein ␤-arrestin-1 may activate a number of downstream kinases, we pretreated in actin bundle formation in the cell body and for cell polarization PMNs with a specific inhibitor of MAPK-activated protein ki- (i.e., pseudopodia formation), we introduced neutralizing Abs to nase-2 (MAPKAPK-2) that may be activated by p38 MAPK and is ␤-arrestin-1 or IgG isotypes controls followed by stimulation of known to elicit actin-mediated cytoskeletal rearrangement (27). PMNs for 1 min with PAF or buffer (Fig. 8). To assess polariza- Inhibition of MAPKAPK-2 caused virtually complete disappear- tion, Nomarski images were obtained to view the formation of ance of the actin bundles in the cell body (Fig. 8C, xv) but had pseudopodia as evidence for active actin rearrangement based on little effect on PAF-induced cell polarization and pseudopod for- the prior results seen in Fig. 7. The formation of actin bundles were mation (Fig. 8C, xiii), similar to PAF-stimulated PMNs (Fig. 8D, assessed using Abs specific to an epitope present only after the i and ii). formation of actin bundles. The spatial relationship with the PAFR was determined using an Ab against an extracellular epitope to the LSP-1 colocalization and regulation of actin bundle formation PAFR, and slides were labeled before permeabilization to ensure Because LSP-1 is a known substrate for MAPKAPK-2 and present extracellular labeling (Fig. 8A). IgG isotype cells treated with PAF in PMNs, whole cell lysates were prepared from PMNs stimulated demonstrated cell polarization (Fig. 8A, v) and actin bundle for- with buffer or 2 ␮M PAF for 1 min (28). Phosphorylated LSP-1 mation (Fig. 8A, vii). Moreover, these actin bundles colocalized demonstrated immunoreactivity following PAF treatment, but was with the PAFR (Fig. 8A, viii). In contrast, intracellular neutraliza- not present in buffer-treated controls (Fig. 9A). Digital microscopy The Journal of Immunology 7045 Downloaded from

FIGURE 7. PAF-mediated actin rearrangement. PMNs stimulated with buffer or PAF were fixed, permeabilized, and Nomarski images (i and iii) were obtained using Nomarski-modified differential interference contrast microscopy. A, Resting cells (i and ii) demonstrated smooth edges and peripheral actin distribution, whereas PAF treatment caused cell polarization (iii) with F-actin distribution occurring at the pseudopod when a plane that transects the http://www.jimmunol.org/ pseudopodia is visualized (iv; white arrow). A more central cross-section of the cell body (v; white arrow) saw more peripheral actin distribution with some actin beginning to form at the edge of the pseudopod (scale bar, 7 ␮m). Additionally, the angle between two actin filaments with the same point of origin was measured, and the polarized edge was magnified to demonstrate an ϳ70° angle (vi). B, Nomarski images show an increase in cell polarization with PAF treatment (iii) compared with resting cell (i) and similar to A. Using an Ab specific for an epitope only found when actin bundles are formed, PMNs had very little actin bundle formation in resting cells (ii), with immunoreactivity increasing with PAF treatment (iv–v). Planes transecting the pseudopodia (iv; white inset) did not demonstrate large increases in actin bundle formation as compared with a plane corresponding to the cell body proper (v; white inset) (scale bar, 7 ␮m). C, The distinction between resting (i) and PAF-treated (iii) Nomarski images and how they correlate to polarization and active actin polymerization cells were visualized for ARP3 (ii and iv; red), which had an even distribution in resting cells with accumulation at the polarized edge ␮ following PAF treatment (scale bar, 7 m). Data in all experiments are representative of three independent experiments. by guest on September 24, 2021

demonstrated that LSP-1 has a peripheral distribution irrespective tion of p38 MAPK elicited two distinct forms of actin polymer- of buffer or PAF treatment (Fig. 9B, i and iv; green); however, ization through stimulation of two separate pathways: 1) ARP2/3 PAF elicited the formation of actin bundles (Fig. 9B, ii and v; red) leading to the formation of the pseudopod, i.e., cell polarization, that colocalized with LSP-1 (Fig. 9B, vi). To study the dependence and 2) MAPKAPK-2 phosphorylation of LSP-1 resulting in actin of actin bundles on LSP-1, Abs specific to the C terminus of LSP-1 bundle in the cell body (28). All of the described signaling steps, or an IgG isotype were introduced. Isotype controls had little effect as well as actin rearrangement, occurred within 60 s of receptor on actin bundle formation, whereas Abs against LSP-1 inhibited ligation, proximal and independent of dynamin-2-mediated endo- actin bundle formation (Fig. 9, C–D). Finally, to understand the somal scission. dependence of PAFR internalization on LSP-1, Abs against LSP-1 In the described experiments, we used primary cells and the were introduced, and the PAFR visualized with an Ab to the ex- intracellular introduction of specific Abs for the neutralization of ternal N terminus. Neutralization of LSP-1 diminished the ability CME proteins, which complemented the data using immunopre- of PAF-mediated receptor internalization, similar to resting cells cipitation and digital microscopy. Our purpose was predicated (Fig. 9, E and F). In contrast, PAF treatment of IgG isotype cells upon the suggestion that translation of findings from immortalized significantly internalized the PAFR as compared with LSP-1 Ab- cell lines may not always hold true for primary cells or in vivo treated cells as well as buffer-treated IgG isotype cells (Fig. 9, E (30). The presented data have some similarities with previous work and F). in transformed cell lines, specifically the roles of ␤-arrestin-1 and dynamin-2 (4, 31). However, our data contrast previous PAFR Discussion models in which ␤-arrestin-1 supports a p42/44 signalosome rather The rapid response of PMNs to extracellular stimuli, especially than a p38 MAPK signalosome (32). These data demonstrate that chemoattractants, is inherent to their defensive function in the tis- in primary cell as compared with immortalized cells, ligation may sues. PAF activation of its receptor on the PMN membrane caused activate distinctly different signaling pathways, though using the CME, as evidenced by the physical association, FRET positive, of same scaffolding. the PAFR with known proteins required for CME (13, 29). More- Previous data have documented that PAF signaling (PAFR) in over, PAFR desensitization was ␤-arrestin-1-dependent, with re- transfected cells requires CME because the observed PAF-induced ceptor internalization requiring both ␤-arrestin-1 and dynamin-2. effects were dynamin-2-dependent and because RNA silencing of This translocation and binding of ␤-arrestin-1 to the PAFR pro- dynmain-2 resulted in inhibition of PAF-induced effects, including vided a platform for recruitment of a p38 MAPK signalosome receptor internalization and degradation (29). Furthermore, PAF (ASK1/MKK3/p38 MAPK) and its subsequent activation. Activa- signaling also required ␤-arrestin-1 (arrestin-2), for mutations of 7046 PAF CME REQUIRES ␤-ARRESTIN IN p38-INDUCED ACTIN BUNDLES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 8. Regulation of actin bundle formation by ␤-arrestin-1, p38 MAPK, and MAPKAPK-2. A, PMNs were loaded with an IgG isotype (i–viii) or a ␤-arrestin-1 Ab (ix–xii) and stimulated with buffer or PAF (1 min). Nomarski images (i, v, and ix) revealed that the ␤-arrestin-1 Ab abrogated cell polarization similar to resting cells, contrasting the PAF-treated cells. Staining for the PAFR (ii, vi, x; green) and actin bundles (iii, vii, xi; red) demonstrated actin bundle formation following PAF treatment, but was abrogated to levels similar to controls when loaded with the ␤-arrestin-1 Ab. The actin bundle formations seen in IgG-isotype PAF-treated cells colocalized with the PAFR (viii) (scale bar, 7 ␮m). Data are representative of three independent experiments. B, The differences in actin bundle formation (i) and cell polarization (ii), determined by the presence of a pseudopod between PAF-treated ,p Ͻ 0.01; Ͼ20 cells in two independent experiments). C ,ءء) cells in both resting and the ␤-arrestin-1 Ab-loaded cells, were statistically different Pretreatment of cells with the p38 MAPK inhibitor (SB203580) or DMSO followed by PAF stimulation (ix and xii) inhibited both cell polarization and actin bundle formation as compared with buffer pretreatment/PAF-treated cells (i–viii). In contrast, pretreatment with a MAPKAPK-2 inhibitory peptide (xiii–xvi) had little effect on cell polarization, though actin bundle formation was diminished (scale bar, 7 ␮m). Data are representative of three independent p Ͻ ,ءء) experiments. D, The differences in actin bundle formation (i) between the inhibitory groups and PAF-treated cells were statistically significant p Ͻ ,ءء) using ANOVA). Results represent the mean Ϯ SEM from Ͼ20 cells in two independent experiments. Significant differences were similar ,0.01 0.01, using ANOVA) in cell polarization (ii) between the SB203850 group and PAF-treated and MAPKAPK-2 inhibitory peptide group. Results represent the mean Ϯ SEM from Ͼ20 cells in two independent experiments. this protein abrogated the effects of PAF in this immortalized cell its ability to support the assembly of signalosomes specifically line (13). Similar to these data, PAF ligation of its receptor on related to cell function remains undefined (13, 29, 32). In this PMNs rapidly initiated CME as evidenced by the physical asso- study, we describe new insights into the assembly of signaling ciation of the PAFR with the clathrin H chain, the ␣-adaptin sub- cascades in response to receptor ligation before dynamin-2-in- unit of the AP-2 domain of the clathrin pit, and dynamin-2, pro- duced vesicle scission. The role of members of the arrestin family teins integral to CME (33, 34). In addition, PAF stimulation of to mediate kinase activation is also a well-established phenome- PMNs resulted in the internalization of the PAFR, which was dy- non, including activation of members of the Src family, ERK1/2, namin-2- and ␤-arrestin-1-dependent, consistent with previous re- and JNK3 (2, 4, 6). Activation often includes the recruitment and ports showing dynamin mutants caused attenuation of endogenous interaction of arrestins with upstream MAPKs (ASK1, MEK3, receptor recycling (29). MKK3, and MEK kinase-2) (31, 35–37). Our data demonstrated Although the PAFR is well characterized with respect to arrestin that ASK1 is the MAP3K recruited to ␤-arrestin-1, which was binding for the termination of heterotrimeric G protein signaling, surprising because a number of investigators have postulated that The Journal of Immunology 7047 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 9. LSP-1 colocalization and regulation of actin bundle formation. A, PMNs were stimulated with buffer or PAF for 1 min, and whole cell lysates prepared, which demonstrated immunoreactivity with an Ab speciﬁc to LSP-1 phosphorylation (p-LSP-1) following PAF treatment. B, Slides were prepared and Abs against LSP-1 (i and iv) and actin bundles (ii and v) showed colocalization following PAF treatment (vi), which was not present in buffer-treated controls (iii) (scale bar, 7 ␮m). Data are representative of three independent experiments. C and D, Neutralization of LSP-1 demonstrated inhibition of actin bundle formation following PAF treatment, with normal formation in PAF-treated IgG isotype cells. Results represent the mean Ϯ SEM from Ͼ20 cells p Ͻ 0.01, using ANOVA. E and F, The differences in actin bundle formation between PAF-treated cells loaded with ,ءء .in two independent experiments p Ͻ 0.01, using ANOVA. Results represent the mean Ϯ SEM; from Ͼ20 ,ءء .LSP-1 neutralizing Abs and IgG isotype Abs were statistically signiﬁcant cells in two independent experiments.

TAK or PAK were the most likely candidates for p38 MAPK demonstrated the physical association (FRET positive) with ␤-ar- activation (TAK/PAK) (38), and ASK1 is best characterized in restin-1 supporting this assembly pattern. apoptosis (39, 40). Under osmotic stress or stimulation with A significant finding in this study is the divergent signaling di- TNF-␣, ASK1 is activated by 1) the oxidation of thioredoxin, re- rectly downstream of p38 MAPK. p38 MAPK activation at the leasing ASK1 and promoting dimerization or 2) the dephosphor- membrane lead to the formation of two disparate types of poly- ylation of Ser967 on ASK1, which causes dissociation from protein merized actin. The first type is the 70° actin filaments in the pseu- 14-3-3 (41–44). In both cases, release of ASK1 leads to p38 dopodia responsible for cell polarization that colocalized with MAPK activation and apoptosis; however, PMNs incubated with ARP2/3 and the second is the actin bundles in the cell body at the PAF demonstrated significantly higher survival rates at 24 h com- site of the PAFR. Antagonism (SB203580) of p38 MAPK signif- pared with controls, and pretreatment of PMNs with PAF abolishes icantly inhibited both cell polarization and actin bundle formation; TNF-␣-induced apoptosis (45). With regard to cassette assembly, whereas, inhibition of MAPKAPK-2 or intracellular neutralization McDonald et al. (37) reported that cotransfection of ␤-arrestin-2 of LSP-1 solely diminished actin bundle formation. These data are and the angiotensin II type 1a receptor into COS-7 cells led to similar to results demonstrating that leukocytes from ASK1Ϫ/Ϫ or ASK1-mediated activation of JNK3. However, in PMNs ASK1 p38 MAPKϪ/Ϫ mice exhibit impaired actin polymerization and participated in a signaling cassette with MKK3 and p38 MAPK. In decreased chemotaxis (46). addition, for assembly of the ASK1/MKK3/p38 MAPK signalo- The formation of actin bundles at the site of the PAFR is novel some, ␤-arrestin-1 recruited MKK3 (FRET positive) and a pre- and intriguing in PMNs, which lack fascin, a protein associated formed ASK1/p38 MAPK pair, present in control PMNs, for p38 with actin bundles in other cell lines (47). We have demonstrated MAPK activation. Importantly, neither ASK1 nor p38 MAPK that the specific actin epitope presented only upon actin bundle 7048 PAF CME REQUIRES ␤-ARRESTIN IN p38-INDUCED ACTIN BUNDLES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 10. Proposed mechanism of PAF-mediated ␤-arrestin-1-dependent p38 MAPK signaling. A, PAF ligation of its receptor (1) causes the recruitment of the heterotrimeric subunits ␣ and ␤␥ (2), resulting in the release of intracellular calcium (3). Following receptor phosphorylation (4), ␤-arrestin-1 is recruited to the receptor (5i), which recruits MKK3 (5ii), and p38 MAPK/ASK1 (5iii) facilitating the activation (phosphorylation) of MKK3 and p38 MAPK. B, Following the assembly of this complex, elements of CME are recruited, including clathrin H chain and L chain and AP-2 (␤-arrestin- 1-interacting protein), which are necessary for receptor internalization. This complex facilitates the activation (phosphorylation) of MAPKAP2K (2), which then phosphorylates LSP-1 (3) leading the formation of actin bundles at the membrane near the receptor (4). C, The receptor is then internalized in a dynamin-2-dependent manner. The Journal of Immunology 7049 formation was 1) at the site of the PAFR in the cell body (as trophil functions through changes in cytosolic calcium. J. Leukocyte Biol. 73: opposed to the pseudopod), 2) colocalized with MAPKAPK-2 and 511–524. 17. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Three- LSP-1, 3) was inhibited by selective antagonists for MAPKAPK-2, dimensional segregation of supramolecular activation clusters in T cells. Nature 4) colocalized with phosphorylated LSP-1, and 5) was diminished 395: 82–86. when LSP-1 was neutralized by intracellular introduction of a spe- 18. Weiner, O. D., G. Servant, M. D. Welch, T. J. Mitchison, J. W. Sedat, and H. R. cific Ab. Furthermore, LSP-1 is a known substrate for MAP- Bourne. 1999. Spatial control of actin polymerization during neutrophil chemotaxis. Nat. Cell Biol. 1: 75–81. KAPK-2 (28) and has homology with villin, an actin bundling 19. Wouters, F. S., P. I. Bastiaens, K. W. Wirtz, and T. M. Jovin. 1998. FRET protein, implying that LSP-1 may have very similar characteristics microscopy demonstrates molecular association of non-specific lipid transfer pro- (48, 49). These results suggest that LSP-1 may serve a similar role tein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J. 17: as fascin in actin bundle formation in PMNs, and future studies are 7179–7189. 20. Perron, A., Z.-G. Chen, D. Gingras, D. J. Dupre´, J. Stao`kova´, and M. Rola- required to corroborate these findings. Pleszczynski. 2003. Agonist-independent desensitization and internalization of In conclusion, PAF ligation of its receptor causes recruitment of the human platelet-activating factor receptor by coumermycin-gyrase B-induced ␤-arrestin-1 to the membrane, which serves a dual role and is dimerization. J. Biol. Chem. 278: 27956–27965. demonstrated in Fig. 10. The data presented provide evidence that 21. Rane, M. J., Y. Pan, S. Singh, D. W. Powell, R. Wu, T. Cummins, Q. Chen, K. R. McLeish, and J. B. Klein. 2003. Heat shock protein 27 controls apoptosis by endocytosis is a complex, multifaceted signaling mechanism that regulating Akt activation. J. Biol. Chem. 278: 27828–27835. may provide multiple signaling platforms to rapidly change the 22. Nick, J. A., N. J. Avdi, S. K. Young, C. Knall, P. Gerwins, G. L. Johnson, and structure and ultimately the function of motile cells, especially G. S. Worthen. 1997. Common and distinct intracellular signaling pathways in leukocytes. Further work is required to establish whether the de- human neutrophils utilized by platelet activating factor and FMLP. J. Clin. Invest. 99: 975–986. Downloaded from scribed signaling platforms are present and conserved in multiple 23. Schaefer, A. W., H. Kamiguchi, E. V. Wong, C. M. Beach, G. Landreth, and V. cell types or whether these transduction pathways inherent to PAF- Lemmon. 1999. Activation of the MAPK signal cascade by the neural cell ad- mediated CME are restricted to leukocytes. hesion molecule L1 requires L1 internalization. J. Biol. Chem. 274: 37965–37973. 24. Benard, O., Z. Naor, and R. Seger. 2001. Role of dynamin, Src, and Ras in the Disclosures protein kinase C-mediated activation of ERK by gonadotropin-releasing hor- The authors have no financial conflict of interest. mone. J. Biol. Chem. 276: 4554–4563. ␤

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