Phosphorylation of Tumor Necrosis Factor Receptor CD120a (p55) by p42 mapk/erk2 Induces Changes in Its Subcellular Localization*

The interaction of tumor necrosis factor-α (TNFα) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42 mapk/erk2 phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207–425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFα, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42 mapk/erk2 and by specific inhibition of p42 mapk/erk2 activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42 mapk/erk2 , and constitutively active MEK-1 followed by metabolic labeling with [32P]orthophosphate indicated that p42 mapk/erk2 phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42 mapk/erk2 -dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.

Tumor necrosis factor-␣ (TNF␣), 1 a pleiotropic cytokine pro-duced mainly by macrophages, regulates many aspects of the inflammatory response and host defense mechanisms against pathogenic microorganisms (1,2). TNF␣ interacts with and induces oligomerization of two cell surface receptors, CD120a (p55) and CD120b (p75), that are collectively responsible for signaling the many distinct cellular responses that are induced by this cytokine (3,4). Following ligand binding, the adapter molecule TRADD binds through its death domain to the death domain of CD120a (p55) (5) thereby forming a platform for the assembly of other signaling molecules including TRAF2 and RIP (6,7). These interactions set in motion the activation of downstream signaling cascades, including the activation of members of the mitogen-activated protein kinase (MAPK) (8) and I-B kinase families (9 -11), culminating in the transactivation of transcription factors, especially AP-1 and NF-B, that regulate the expression of genes involved in the inflammatory response.
Although RIP has been implicated in the initiation of NF-B activation, these findings do not, as yet, invoke other receptorassociated protein Ser/Thr or Tyr kinases in the functions of CD120a (p55). However, several reports have shown protein Ser/Thr kinase activities to be bound by sequences located within the intracellular domain of both TNF receptors (12,13), as well as by the related lymphotoxin-␤ receptor (14). In the case of CD120b (p75), the receptor-associated kinase has been identified as casein kinase I, and its activity has been shown to prevent apoptotic cell death (15). A serine-kinase activity that co-immunoprecipitates with CD120a (p55) in human U937 cells has also been shown to phosphorylate receptor-associated substrates of molecular masses 125, 97, 85, and 60 kDa in a ligand-dependent fashion (16). Darnay and colleagues (12) have employed fusion proteins between glutathione S-transferase (GST) and various truncation and deletion mutants of the cytoplasmic domain of CD120a (p55) to investigate a Ser/Thr kinase activity designated p60 TRAK and have shown that p60 TRAK is capable of phosphorylating the cytoplasmic domain of CD120a (p55) but not that of CD120b (p75). The only CD120a (p55)-associated Ser/Thr-kinase to be cloned is RIP, a death domain-containing receptor-interacting protein (6,17). However, although its kinase activity has been implicated in downstream signaling leading to the activation of I-B kinases (18), it has not been shown to stimulate phosphorylation of CD120a (p55) itself. Although these reports have provided intriguing and important insights into CD120a (p55)-associated kinases and their mode of interaction with the cytoplasmic domain of CD120a (p55), the identity and function(s) of CD120a (p55)-Ser/Thr kinase activities remain largely unknown.
The MAPKs currently comprise three major subfamilies in mammalian cells as follows: 1) the p38 mapk subfamily (19,20); 2) the extracellular signal-regulated kinases (ERK) p44 mapk/erk1 and p42 mapk/erk2 (21); and 3) the c-Jun NH 2 -terminal kinases (JNK) which consist of multiple isoforms of both p46 jnk1 and p54 jnk2 (22,23). All family members are prolinedirected Ser/Thr kinases, and while p38 mapk and JNK subfamily members appear to phosphorylate the basic kinase consensus motif, (S/T)P (24), ERK-dependent phosphorylation events are directed toward the consensus sequence PX 1,2 (S/ T)P, where X represents any non-acidic residue (25,26). In work previously reported from this laboratory, we have shown that specific members of each MAPK subfamily are rapidly and transiently activated in response to stimulation of mouse macrophages with TNF␣ (27)(28)(29). However, although the targets of these kinases include transcription factors involved in the inflammatory response, we have questioned if they also include CD120a (p55) itself. The 218 amino acids of the cytoplasmic domain of mouse CD120a (p55) are organized into the death domain (residues 326 -411) (30) and a Ser-, Thr-, and Pro-rich membrane proximal region broadly located between residues 230 and 280. Within this region are seven basic Prodirected kinase motifs and four sequences bearing the consensus ERK phosphorylation motif. Based on these collective observations we have investigated (i) if MAPKs interact with and phosphorylate the cytoplasmic domain of CD120a (p55) and (ii) the consequence of this event on CD120a (p55) subcellular localization.
Macrophage Isolation, Culture, and Labeling-Monolayers of mouse bone marrow-derived macrophages were prepared from femoral and tibial bone marrow as described previously (27). To label the endogenous CD120a (p55), bone marrow-derived macrophages (ϳ2 ϫ 10 6 cells) were washed with phosphate-free medium and incubated with 1 mCi of [ 32 P]orthophosphate in phosphate-free Eagle's minimal essential medium for 6 h. Cells were stimulated with 1 M okadaic acid during the last 3 h of the incubation period, and lysed in 1 ml of Nonidet P-40 buffer. Lysates were treated with 5 units of DNase for 15 min at 4°C, before being pre-cleared with 15 l of protein A/G-Plus Sepharose beads. CD120a (p55) was then immunoprecipitated overnight at 4°C with 2.5 g of anti-CD120a (p55) antagonist hamster monoclonal antibody or 2.5 g of H57 anti-T-cell receptor hamster monoclonal antibody as a control, and 25 l of protein A/G-Plus-Sepharose beads. The beads were washed 12 times with Nonidet P-40 buffer. Duplicates of each condition were pooled and resolved by SDS-PAGE, transferred onto nitrocellulose, and subjected to autoradiography.
Construction of Expression Vectors-The GST-CD120a-(207-425) fusion protein was created by fusing the COOH terminus of glutathione S-transferase (GST) to the full-length intracellular domain (amino acids 207-425) of mouse CD120a (p55). A 654-base pair EcoRI/SalI cDNA fragment encoding these residues was amplified by polymerase chain reaction (PCR), digested with EcoRI and SalI, and ligated into EcoRI/ SalI-digested pGEX-5X-1. The cDNA encoding amino acids 1-425 (i.e. the entire receptor except for the signal peptide) was amplified by PCR, digested with EcoRI, and subcloned into EcoRI-digested pFLAG-CMV-1 downstream of the signal sequence and the sequence encoding the FLAG epitope (GYKDDDDK). CD120a (p55) T236A, S240A, S244A, S270A and CD120a (p55) T236D, S240E, S244E, S270E mutants were constructed using overlapping PCR and also ligated into pFLAG-CMV-1. The fidelity of all the constructs was verified by restriction enzyme analysis and nucleotide sequencing. The expression vectors for HA-tagged Xenopus p42 mapk/erk2 (HA.ERK2.WT) and a constitutively active mutated form of Xenopus MEK-1 (MEK1.CA) in the pcDNA3 vector (Invitrogen, Carlsbad, CA) were a generous gift from Dr. Lynn Heasley, Department of Renal Medicine, University of Colorado Health Sciences Center, Denver, CO. The vector expressing VSV-G bearing a COOH-terminal KKTN motif was kindly provided by Dr. Alexander van der Bliek, Los Angeles, CA.
Expression and Purification of GST Fusion Proteins-The expression and purification of GST and the GST-CD120a-(207-425) fusion protein were carried out as described (31). The amount of fusion protein was estimated by SDS-PAGE under reducing conditions using varying amounts of bovine serum albumin as a standard, followed by staining with Coomassie Blue.
Separation of Kinase Activities by Fast Protein Liquid Chromatography-Unstimulated or TNF␣-stimulated macrophage monolayers (ϳ5 ϫ 10 7 cells) were scraped into 2 ml of ice-cold lysis buffer (50 mM Tris buffer, pH 8.0, containing 1% Nonidet P-40, 50 mM NaCl, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 5 g/ml aprotinin, and 1 mM Na 3 VO 4 ). Nuclear material was pelleted by centrifugation at 14,000 ϫ g for 10 min at 4°C. The supernatant was loaded onto an anion-exchange column (Mono-Q, BioLogic Systems, Inc.) equilibrated in 50 mM ␤-glycerophosphate, pH 7.2, containing 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and 0.05 M NaCl. The elution profile consisted of a flow rate of 1 ml/min for 40 ml with a linear gradient of 0.05 to 0.2 M NaCl in equilibration buffer, followed by a flow rate of 1 ml/min for 4 ml with a linear gradient of 0.2 to 1 M NaCl, then by an isocratic flow of 1 M NaCl at a rate of 1 ml/min for 20 ml. Two ml fractions were collected, and 1-ml aliquots were tested for in vitro kinase activity using GST-CD120a-(207-425) beads as substrate as described below.
In Vitro Binding of Macrophage Lysates to GST-CD120a-(207-425)-Macrophage monolayers (ϳ2 ϫ 10 6 cells) were rinsed with ice-cold 20 mM Hepes-buffered saline, pH 7.4, and lysed on ice in 500 l of Nonidet P-40 lysis buffer (50 mM Tris buffer, pH 8.0, containing 1% Nonidet P-40, 137 mM NaCl, and protease inhibitors as described above). Nuclear material was pelleted by centrifugation at 14,000 ϫ g for 10 min at 4°C. Supernatants were transferred to a new tube and precleared with 20 l of washed GST-coated beads for 20 min at 4°C on a rotator. The beads were spun down at 3000 rpm for 3 min, the supernatants were transferred to new tubes, and incubated with 20 l of washed GST-CD120a-(207-425)-coated beads for 150 min at 4°C on a rotator. After incubation, the beads were spun down at 3000 rpm for 3 min, washed twice with Nonidet P-40 lysis buffer, and then twice with PAN buffer (10 mM Pipes buffer, pH 7.0, containing 100 mM NaCl and 10 g/ml aprotinin). After the last wash, the beads were resuspended in 40 l of PAN buffer to bring the mixture up to 60 l and then used in the in vitro kinase assay as described below. In experiments in which the binding of cytosolic proteins to GST-CD120-(207-425) was determined by Western blotting, ϳ5 ϫ 10 7 cells were lysed and prepared as described above before being directly subjected to SDS-PAGE and transferred to nitrocellulose membranes.
In Vitro Kinase Assays-Kinase assays were performed at 30°C for 30 min in kinase buffer (10 mM Pipes buffer, pH 7.2, containing 10 mM MnCl 2 and 20 g/ml aprotinin) together with 10 Ci of [␥-32 P]ATP. The kinase reactions were terminated by the addition of 20 l of 5ϫ Laemmli sample buffer containing 100 mM dithiothreitol. The mixtures were boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose for autoradiography and Western analysis. In in vitro kinase assays with recombinant p42 mapk/erk2 , 50 ng of recombinant MEK-1-activated p42 mapk/erk2 (i.e. constitutively active) were incubated for 150 min at 4°C with 20 l of washed GST-CD120a-(207-425) beads resuspended in 40 l of PAN buffer. The kinase reactions were conducted directly or after the beads were washed 4 times with Nonidet P-40 lysis buffer and 4 times with PAN buffer as described above.
Immune Complex Kinase Assays and Immunodepletion-Mouse macrophages were lysed as described above, and post-nuclear supernatants were precleared with 15 l of protein A-Sepharose beads. Individual MAPKs were immunoprecipitated by incubation at 4°C for 2 h with 5 g of p38 mapk , p42 mapk/erk2 , and p46 jnk1 antibodies and 25 l of protein A-Sepharose beads. The beads were then washed twice with Nonidet P-40 lysis buffer and twice with PAN buffer. Kinase activity was assayed as described above with either the GST-CD120a-(207-425) fusion protein or recombinant ATF-2 as substrate.
For immunodepletion experiments, the cells were lysed as described above. The supernatants were precleared with 15 l of protein A-Sepharose beads, and the MAPKs were immunoprecipitated by incubation at 4°C for 2 h with 5 g of p38 mapk , p42 mapk/erk2 , and p46 jnk1 antibodies or non-immune rabbit IgG, and 25 l of protein A-Sepharose beads. The beads were then spun down at 3000 rpm for 3 min, and the supernatants were transferred to a new tube, precleared with 20 l of washed GST beads for 20 min at 4°C, and incubated with 20 l of washed GST-CD120a-(207-425) beads for 150 min at 4°C on a rotator. After incubation, an in vitro kinase assay was conducted as described above.
Transfections and in Vivo Labeling with [ 32 P]Orthophosphate-COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, and 2 mM glutamine. ϳ2 ϫ 10 5 COS-7 cells/well were seeded in 6-well (35 mm) dishes and grown in 7.5% CO 2 . Cells were transfected the following day using the LipofectAMINE TM reagent (Life Technologies, Inc.) according to the recommendations of the manufacturer. 1.5 g of DNA were transfected with 10 l of LipofectAMINE TM reagent in a total volume of 1 ml of Opti-MEM® I reduced serum medium (Life Technologies, Inc.). The amount of transfected DNA was kept constant with pcDNA3 empty vector. Eighteen h after transfection, the cells were washed with phosphate-free medium and metabolically labeled by incubation in phosphate-free Eagle's minimal essential medium containing 1 mCi of [ 32 P]orthophosphate for 6 h. At the end of the incubation period, the cells were lysed in 250 l of Nonidet P-40 buffer, and post-nuclear supernatants were precleared with 25 l of protein A-Sepharose beads. CD120a (p55) was immunoprecipitated overnight at 4°C with 10 g of anti-CD120a (p55) antagonist monoclonal antibody and 50 l of protein A-Sepharose beads. The beads were washed 12 times with Nonidet P-40 lysis buffer and resolved by SDS-PAGE.
Confocal Immunofluorescence Microscopy-ϳ10 5 HeLa cells were seeded in 12-well plates containing 18-mm glass coverslips and grown in 5% CO 2 . Cells were transfected with 100 ng of DNA the following day using the LipofectAMINE TM reagent (Life Technologies, Inc.). Fourteen h after transfection, the cells were washed with PBS, fixed for 15 min at room temperature in a solution containing 3% (w/v) paraformaldehyde and 3% (w/v) sucrose in PBS, pH 7.5, washed again, and permeabilized with 0.2% (v/v) Triton X-100 for 10 min. The cells were then washed, blocked for 30 min in Hank's balanced solution containing 10% normal donkey serum, and then incubated with the primary antibodies (1:100) in blocking solution for 2 h. After washing with PBS, the cells were incubated with Cy3-conjugated donkey anti-goat or Armenian hamster IgG and/or fluorescein-conjugated donkey anti-mouse IgG (1:200). The coverslips were washed with PBS, incubated overnight in PBS supplemented with 0.02% sodium azide, and mounted in a solution containing 90% glycerol, 10% Tris-HCl, pH 8.5, and 20 mg/ml o-phenylenediamine as an anti-fading agent. To stain the endogenous CD120a (p55), HeLa cells were stimulated with 0.1 M okadaic acid for 150 min, transfected with 100 ng of DNA for 24 h, or left untreated. Cells were fixed and permeabilized as above, before being blocked for 60 min in Hank's balanced solution containing 5% normal donkey serum and 5% normal goat serum. Cells were then incubated for 2 h with the primary antibodies in blocking buffer (1:200 of anti-CD120a (p55) hamster monoclonal antibody or 1:200 of H57 anti-T-cell receptor hamster monoclonal antibody as a control). Cells were washed with PBS, incubated for 1 h with Cy3-conjugated goat anti-hamster IgG (1:200 in blocking buffer), washed, and then incubated for 1 h with Cy3-conjugated donkey antigoat IgG (1:200 in blocking buffer). The coverslips were washed, incubated overnight in PBS, and mounted. To visualize the endoplasmic reticulum, cells were incubated with 5 g/ml Alexa 488-conjugated concanavalin A (Molecular Probes, Eugene, OR) together with the secondary antibodies. The specificity of the secondary antibodies was checked to make sure they recognized only the appropriate primary antibody. DNA was stained with the Hoechst dye 33342 at 10 g/ml. Cells were observed with a Leica DMR/XA confocal immunofluorescence microscope using a 100 ϫ Plan objective. Digital images were captured using a SensiCam camera, deconvoluted using the software "Slidebook" (Intelligent Imaging Innovations, Inc., Denver, CO) to remove out of focus fluorescence, and processed using Adobe Photoshop 4.0 (Adobe Systems, Inc.).
Electron Microscopy-HeLa cell monolayers grown on Thermanox coverslips were fixed in 4% (w/v) paraformaldehyde and 0.05% (v/v) glutaraldehyde, permeabilized in 0.2% (v/v) Triton X-100, and blocked in 10% normal rabbit serum for 10 min at room temperature. The coverslips were incubated with goat anti-CD120a (p55) (1 g/ml in blocking buffer) for 2 h at room temperature. After rinsing, bound antibody was detected using the Vector ABC Elite kit following the manufacturer's instructions. The coverslips were then post-stained with osmium tetroxide and uranyl acetate, dehydrated with graded solutions of ethanol, and embedded in plastic. Ultrathin sections were cut on a Reichert ultratome and examined in a Philips CM10 electron microscope at 80 kV.

RESULTS
A Kinase Activity That Binds to and Phosphorylates CD120a-(207-425) Co-elutes with p42 mapk/erk2 -The TNF receptor CD120a (p55) is expressed in low abundance on all cell types studied, and macrophages are no exception (32). Therefore, to study the phosphorylation of CD120a (p55) we fused residues 207-425 which encode the entire intracellular domain of mouse CD120a (p55) to GST and expressed the GST-CD120-(207-425) fusion protein in Escherichia coli. The fusion protein was then bound to glutathione-Sepharose beads and was used to affinity purify receptor-interacting proteins from lysates of unstimulated and TNF␣-stimulated mouse macrophages. Kinase(s) capable of phosphorylating GST-CD120a-(207-425) were then detected in an in vitro kinase assay. As can be seen in Figs. 2 and 3, incubation of mouse macrophages with TNF␣ (10 ng/ml for 10 min) led to the activation of a kinase activity that bound to and trans-phosphorylated the GST-CD120a-(207-425) fusion protein but not GST alone. The major phosphoprotein of ϳ51 kDa co-localized with the GST-CD120a-(207-425) fusion protein. An additional phosphoprotein of retarded mobility, believed to be a multiply phosphorylated species of GST-CD120a-(207-425), was also detected.
To begin to explore the possibility that p38 mapk , p42 mapk/erk2 , or p46 jnk1 contributed to the phosphorylation of GST-CD120a-(207-425), we separated these MAPKs from lysates of TNF␣stimulated (10 ng/ml for 10 min) and unstimulated macrophages by fast protein liquid chromatography over a Mono-Q anion-exchange column and determined if CD120a-(207-425) kinase activity co-eluted with either p38 mapk , p42 mapk/erk2 , or p46 jnk1 . As can be seen in Fig. 1B, three peaks of kinase activity were detected in response to TNF␣ stimulation, and only two peaks were detected in lysates from unstimulated cells. The TNF␣-inducible GST-CD120a-(207-425) kinase activity (peak 2) co-eluted with phosphorylated p42 mapk/erk2 as shown by immunoblotting with an antibody that recognized both phosphorylated p42 mapk/erk2 and p44 mapk/erk1 (Fig. 1D). It can also be seen that p42 mapk/erk2 eluted at approximately 0.17 M NaCl and was separated from p44 mapk/erk1 . Peak 1 kinase activity eluted at the beginning of the linear NaCl gradient and did not co-localize with any of the MAPKs, whereas peak 3 kinase activity eluted at the beginning of the isocratic flow of 1 M NaCl and co-eluted with p38 mapk (and probably other kinases) during this high salt wash.
p42 mapk/erk2 Phosphorylates GST-CD120a-(207-425) in Vitro-Based on the co-elution of CD120a-(207-425) kinase activity and p42 mapk/erk2 , we next questioned if the TNF␣induced phosphorylation of GST-CD120a-(207-425) was dependent upon p42 mapk/erk2 by blocking its activation with a pharmacologic antagonist of MEK activation, PD098059 (33). Macrophage monolayers were pretreated with PD098059 (30 M) for 1 h and then incubated with TNF␣ (10 ng/ml) for 10 min in the continued presence of the antagonist before being lysed and subjected to an in vitro kinase assay using GST-CD120a-(207-425)-coated Sepharose beads as substrate. As can be seen in Fig. 2A, pretreatment with PD098059 completely inhibited the induction of GST-CD120a-(207-425) kinase activity in response to TNF␣. In contrast, pretreatment of macrophage monolayers with SB203580, a competitive inhibitor of p38 mapk , had no effect on the phosphorylation of the GST-CD120a-(207-425) fusion protein. To verify that the inhibition of GST-CD120a-(207-425) kinase activity by PD098059 was not due to inhibition of other protein kinases that may act upstream or downstream of p42 mapk/erk2 , including phosphatidylinositol-3Ј-OH kinase or p70 S6 kinase, we pretreated macrophages with wortmannin, an inhibitor of phosphatidylinositol-3Ј-OH kinase, (34) and rapamycin, a specific inhibitor of the p70 S6 kinase (35) prior to incubation with TNF␣. Neither inhibitor had any effect on the phosphorylation of the GST-CD120a-(207-425) fusion protein ( Fig. 2A).
To establish directly a role for p42 mapk/erk2 in the phosphorylation of GST-CD120a-(207-425), p38 mapk , p42 mapk/erk2 , and p46 jnk1 were immunoprecipitated from detergent lysates of TNF␣-stimulated macrophages (10 ng/ml for 10 min), and residual GST-CD120-(207-425) kinase activity was quantified in the post-immunoprecipitation supernatants. Immunodepletion with anti-p42 mapk/erk2 antibodies markedly, although incompletely, removed p42 mapk/erk2 even when combinations of polyclonal antibodies were used (Fig. 2B, lower panel). Nevertheless, the majority of the TNF␣-inducible kinase activity was depleted under the conditions used (Fig. 2B, upper panel). Quantitative immunodepletion was achieved with anti-p38 mapk and anti-p46 jnk1 antibodies (Fig. 2B, lower panel) but had no effect on the TNF␣-induced phosphorylation of the GST-CD120a-(207-425) fusion protein (Fig. 2B, upper panel). Conversely, and as shown in Fig. 3A, immunoprecipitated p42 mapk/erk2 , but not p38 mapk or p46 jnk1 , was found to efficiently phosphorylate the GST-CD120a-(207-425) fusion protein in vitro, even though all three immunoprecipitated MAPKs were catalytically active, as illustrated by their ability to phosphorylate ATF-2 (Fig. 3A). The ability of p42 mapk/erk2 to phosphorylate the GST-CD120a-(207-425) was also confirmed using purified constitutively active recombinant p42 mapk/erk2 Macrophage monolayers were pretreated with the MEK-1 inhibitor PD098059 (30 M in dimethyl sulfoxide (DMSO)), the phosphatidylinositol-3Ј-OH kinase (PI3ЈK) inhibitor wortmannin (10 M in dimethyl sulfoxide), the p70 S6 kinase pathway inhibitor rapamycin (10 g/ml in ethanol), the p38 mapk competitive inhibitor SB203580 (1 M in dimethyl sulfoxide), dimethyl sulfoxide, or ethanol, and treated with TNF␣ (10 ng/ml for 10 min). The cells were lysed as described and lysates were subjected to an in vitro kinase assay using GST-CD120a-(207-425) beads or control GST beads as substrate. B, immunodepletion of p42 mapk/erk2 suppresses the TNF␣-induced kinase activity associated with the GST-CD120a-(207-425) fusion protein. The three MAPKs were immunoprecipitated from detergent lysates of unstimulated or TNF␣-stimulated (10 ng/ml, 10 min) macrophages using specific antibodies. The residual kinase activity was measured in the post-immunoprecipitation (Post-IP) lysates in an in vitro kinase assay as described above. Pre-(Pre-IP) and post-immunoprecipitation lysates were subjected to Western blotting with specific anti-MAPK antibodies as indicated. (Fig. 3B). We also conducted experiments in which the constitutively active recombinant p42 mapk/erk2 was allowed to bind to the GST-CD120a-(207-425)-coated beads. Unbound kinase was then removed by extensively washing the beads before conducting the in vitro kinase reaction. As can be seen in Fig.  3B, phosphorylation of the GST-CD120a-(207-425) fusion protein was detected under these conditions, albeit with somewhat reduced efficiency (ϳ50%), indicating that p42 mapk/erk2 was bound to the GST-CD120a-(207-425) fusion protein in the absence of adapter or other signaling molecules.
Binding of p42 mapk/erk2 to the GST-CD120a-(207-425) Fusion Protein-The purification of the receptor-associated kinase activity initially involved affinity adsorption of receptorinteracting proteins to the GST-CD120a-(207-425) fusion protein, thus establishing that p42 mapk/erk2 must bind to the fusion protein to be detected. Given the findings described above, we next investigated the ligand dependence and specificity of this interaction in lysates from unstimulated and TNF␣-stimulated macrophages. Macrophage lysates were incubated with GST-CD120a-(207-425) fusion protein-coated beads. The beads were then washed, and the bound proteins were separated by SDS-PAGE. Western blotting showed that both p42 mapk/erk2 and p44 mapk/erk1 , but not p38 mapk or p46 jnk1 , were bound to the GST-CD120a-(207-425) fusion protein (Fig.  4). Moreover, binding of p42 mapk/erk2 and p44 mapk/erk1 was detected in lysates from both unstimulated and TNF␣-stimulated macrophages suggesting that both inactive and catalytically active members of the ERK family of MAPKs were capable of interacting with the fusion protein. None of the MAPKs were found to bind to GST-coated Sepharose beads.
Phosphorylation of GST-CD120a-(207-425) by Alternative p42 mapk/erk2 Activators-Phosphorylation and activation of p42 mapk/erk2 have been described in response to a broad range of stimuli including cytokines, growth factors, and, in the case of macrophages, certain phagocytic stimuli (36). p38 mapk and p46 jnk1 are activated under stress conditions and in response to a variety of extracellular stimuli including bacterial lipopo-lysaccharide, hyperosmolarity, as well as pro-inflammatory cytokines (19,37). We suspected that since p42 mapk/erk2 is activated by a variety of other cell surface receptors and activated oncogenes, phosphorylation of CD120a (p55) would be expected to be induced in a broader sense by other activators of p42 mapk/erk2 . Such a finding would have important implications for the cross-regulation of CD120a (p55) by other receptors and signal transduction cascades. As can be seen in Fig. 5, exposure of mouse macrophages to the phagocytic stimulus, zymosan particles, stimulated the phosphorylation of the GST-CD120a-(207-425) fusion protein (Fig. 5A) and, as expected, activated p42 mapk/erk2 , p38 mapk , and p46 jnk1 (Fig. 5B). Furthermore, incubation of macrophage monolayers with granulocyte macrophage-CSF and CSF-1 induced the phosphorylation of the GST-CD120a-(207-425) fusion protein. However, although these growth factors induced phosphorylation of p42 mapk/erk2 , they failed to stimulate phosphorylation of p38 mapk or p46 jnk1 . In contrast, incubation of macrophages with (i) hyperosmolar concentrations of sodium chloride or sorbitol or (ii) anisomycin induced activation of p38 mapk and p46 jnk1 but not p42 mapk/erk2 (Fig. 5B). However, these stress-inducing stimuli failed to induce the phosphorylation of the GST-CD120a-(207-425) fusion protein (Fig. 5A). These findings thus indicate that the phosphorylation of sequences in the intracellular domain of CD120a (p55) by p42 mapk/erk2 occurs after activation of p42 mapk/erk2 by a variety of cell surface receptors and thus is not simply a response to TNF␣.
Phosphorylation of CD120a (p55) p42 mapk/erk2 in COS-7 Cells-Collectively, the findings presented support the notion that (i) p42 mapk/erk2 is capable of binding to the intracellular domain of CD120a (p55) and (ii) following activation, p42 mapk/erk2 trans-phosphorylates sites within the intracellular domain of CD120a (p55). However, since these conclusions were based on data obtained using a fusion protein encoding the intracellular domain of CD120a (p55), they raise the question of whether p42 mapk/erk2 is capable of phosphorylating CD120a (p55) in vivo. To address this question, we employed a transfection system in which COS-7 cells were co-transfected with expression vectors encoding CD120a (p55), HA-tagged wild-type p42 mapk/erk2 , and a constitutively active mutant of MEK-1 to drive activation of the transfected p42 mapk/erk2 . Eighteen h after transfection, the cells were labeled with  [ 32 P]orthophosphate for 6 h and lysed as described under "Experimental Procedures." CD120a (p55) was then immunoprecipitated, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and subjected to autoradiography and immunoblotting. As can be seen in Fig. 6B, immunoblotting with an anti-CD120a (p55) antibody showed that CD120a (p55) was expressed at similar levels following transfection into COS-7 cells. Fig. 6A shows that 32 P-labeled CD120a (p55) was detected in COS-7 cells that were co-transfected with vectors encoding CD120a (p55), wild-type p42 mapk/erk2 , and constitutively active MEK-1. Phosphorylation of CD120a (p55) was not detected in cells that were co-transfected with CD120a (p55) and the wild-type p42 mapk/erk2 plasmids in the absence of the constitutively active MEK-1 plasmid (Fig. 6A) nor was it detected in cells transfected with CD120a (p55) and the constitutively active MEK-1 plasmid in the absence of p42 mapk/erk2 (data not shown). Immunoblotting of whole cell lysates obtained from the same transfections with an anti-HA antibody showed that the HA-tagged p42 mapk/erk2 was expressed as predicted (Fig. 6C) and underwent the characteristic mobility shift when COS-7 cells were also transfected with the constitutively active MEK-1 (Fig. 6C). Interestingly, immunoblotting with anti-CD120a (p55) antibodies also revealed a mobility shift in the receptor when cells were co-transfected with expression plasmids encoding CD120a (p55), p42 mapk/erk2 , and constitutively active MEK-1. The gel-shifted CD120a (p55) detected in these immunoblots co-localized with the 32 P-labeled receptor shown in Fig. 6A. These findings demonstrate that activated p42 mapk/erk2 is capable of trans-phosphorylating the cytoplasmic domain of CD120a (p55) in intact cells.
Effect of Phosphorylation by p42 mapk/erk2 on the Subcellular Localization of the CD120a (p55)-The finding that p42 mapk/erk2 phosphorylates CD120a (p55) both in vitro and in intact cells raises the question of the functional consequences of this event. Phosphorylation of receptors modifies their functions in many ways including altering their subcellular localization. To address this issue we studied the effect of phosphorylation of CD120a (p55) on the subcellular localization of the receptor in HeLa cells transfected with expression vectors encoding CD120a (p55). Fourteen h after transfection, the cells were fixed, permeabilized, stained for CD120a (p55), and observed by confocal fluorescence microscopy. As shown in Fig. 7 (A1-2), transfected cells consistently showed a punctate and membranous staining of the receptor as previously reported (58) as well as a localized juxtanuclear staining. To ensure that the punctate staining of CD120a (p55) was localized on the plasma membrane, transfected cells were stained for CD120a (p55) in the absence of detergent permeabilization. CD120a (p55) was still present as a peripheral staining on the plasma membrane (Fig. 7, A3), although tubulin, used as a control to establish the absence of permeabilization, was not stained (data not shown). We suspected that the juxtanuclear staining may represent CD120a (p55) associated with the Golgi apparatus as previously reported (38). When transfected HeLa cells were co-stained for CD120a (p55) and the Golgi marker AP-1, the localized juxtanuclear staining of CD120a (p55) co-localized with AP-1 (Fig. 7, A4 -6), confirming that a significant amount of the receptor is present in the Golgi apparatus.
To determine if phosphorylation of CD120a (p55) altered its subcellular localization, HeLa cells were co-transfected with expression vectors encoding CD120a (p55), wild-type p42 mapk/erk2 , and a constitutively active mutant of MEK-1 and stained for CD120a (p55) and MEK-1. As can be seen in Fig. 7  (B1-3 and C1-3), the punctate membranous expression of CD120a (p55) had disappeared in cells expressing both CD120a (p55) and MEK-1, and the receptor was consistently present in curvilinear tubular structures, which predominated in the pe-  8 -10) and then stimulated with granulocyte-macrophage colony-stimulating factor (10 ng/ml, 10 min), CSF-1 (1000 units/ml), or with a CSF-1-containing medium derived from the culture of L929 cells (10 min). A, the soluble fraction of the lysates was subjected to an in vitro kinase assay as described above. B, the lysates were also separated by SDS-PAGE, transferred on nitrocellulose, and Western-blotted with phosphospecific anti-MAPK antibodies as indicated.

FIG. 6. CD120a (p55) is phosphorylated by catalytically active p42 mapk/erk2 in vivo.
A, COS-7 cells were transfected with the indicated expression vectors, metabolically labeled with [ 32 P]orthophosphate and lysed, and CD120a (p55) was immunoprecipitated. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography. B, the immunoprecipitates were Westernblotted with anti-CD120a (p55) antibodies. C, the lysates were separated by SDS-PAGE, transferred onto nitrocellulose, and subjected to Western blotting with an anti-HA antibody. P-CD120a (p55) and P-p42 mapk/erk2 represent the gel-shifted position of phosphorylated forms of CD120a (p55) and p42 mapk/erk2 , respectively. rinuclear region of these cells. These structures were not stained in non-permeabilized cells, indicating their intracellular location (data not shown) and did not co-localize with the Golgi marker AP-1 (Fig. 7, B4-6 and C4 -6). In addition, the same pattern of staining was seen when the cells were fixed with methanol instead of paraformaldehyde excluding the possibility of a fixation artifact.
These data suggest that the phosphorylation of CD120a not to the phosphorylation of the receptor per se, the four potential ERK phosphorylation sites were mutated to alanine residues (CD120a (p55)T236A, S240A, S244A, and S270A or "p55 4A"). Transfection of the 4-alanine mutant receptor into HeLa cells resulted in a pattern of localization that was similar to the wild-type receptor (Fig. 7, D1-6; D3 shows cells that were not permeabilized). However, co-transfection of the 4-alanine mutant receptor along with p42 mapk/erk2 and constitutively active MEK-1 dramatically reduced the expression of the curvilinear tubular structures as compared with the wild-type receptor (Fig. 7, E1-6). In contrast, when the four potential ERK phosphorylation sites were mutated to Asp and Glu residues to mimic the phosphorylated Ser and Thr residues (CD120a (p55)T236E, S240D, S244D, and S270D or "p55 4D/ E"), the mutant receptor showed a similar curvilinear tubular intracellular localization when transfected in the absence of kinases (Fig. 7, F1-3 and G1-3) to that seen when the wild-type receptor was co-transfected with constitutively active MEK and p42 mapk/erk2 . In addition, the p55 4D/E mutant receptor was not expressed at the plasma membrane and did not co-localize with the Golgi marker AP-1 (Fig. 7, F4 -6 and G4 -6).
To establish the identity of the organelle(s) containing or associated with the phosphorylated receptor, HeLa cells transfected with the p55 4D/E mutant were stained for CD120a (p55) itself and with various dyes and markers specific for the following: (i) the Golgi apparatus (AP-1, wheat germ agglutinin); (ii) actin filaments (phalloidin); (iii) mitochondria (Mitotracker orange); (iv) endosomes (fluorescein-conjugated dextran); (v) lysosomes (antibody for cathepsin D); (vi) vimentin intermediate filaments (anti-vimentin antibodies); (vii) microtubules (antibody for ␣-tubulin); (viii) stable microtubules (antibody for acetyl-tubulin or treatment for 1 h with nocodazole and staining for ␣-tubulin); (ix) the plasma membrane (wheat germ agglutinin); (x) DNA (Hoechst dye 33342); and (xi) kinesin (anti-kinesin antibodies). As shown in Fig. 8, none of these organelles and structures co-localized with the mutated receptor. However, the endoplasmic reticulum, stained with Alexa 488-conjugated concanavalin A, encompassed curvilinear structures that partially co-localized with the p55 4D/E mutant receptor (Fig. 9, A-H). To investigate further the possible association of the phosphorylated receptor with elements of the endoplasmic reticulum, HeLa cells were co-transfected with expression vectors encoding the p55 4D/E receptor and the vesicular stomatitis virus glycoprotein bearing a COOH-terminal KKTN endoplasmic reticulum targeting motif (VSV-G). As shown in Fig. 9 (I-P), the mutated receptor partially co-localized with the VSV-G protein, demonstrating that the intracellular curvilinear and perinuclear tubular structures in which the receptor is present represent elements of the endoplasmic reticulum. As can also be seen in Fig. 9, the elements of the endoplasmic reticulum containing the p55 D/E receptor appeared swollen compared with areas of the endoplasmic reticulum from which the receptor was absent. However, we should emphasize that the phosphorylated receptor was localized in certain areas of the endoplasmic reticulum rather than being uniformly distributed throughout the entire endoplasmic reticulum. Thus, much of the endoplasmic reticulum does not contain the phosphorylated receptor, but most of the phosphorylated receptor is associated with the endoplasmic reticulum. To confirm further the association of the phosphorylated receptor with the endoplasmic reticulum, we transfected HeLa cells with the p55 D/E mutant and subjected the fixed and permeabilized cells to immunochemistry for CD120a (p55) at the electron microscope level. As can be seen in Fig. 10, abundant peroxidase reaction product was detected in structures that bear morphologic identity to the rough endoplasmic reticulum, thus confirming the presence of the phosphorylated receptor in elements of the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of CD120a (p55) by p42 mapk/erk2 abolishes both its plasma membrane and Golgi localization and targets the receptor to tubular elements of the endoplasmic reticulum.
Phosphorylation and Subcellular Redistribution of CD120a (p55) in Intact Cells-To establish the physiological relevance of these observations, we investigated if endogenous CD120a (p55) is phosphorylated in intact macrophages upon ERK activation and the effect of phosphorylation on the redistribution of the receptor to intracellular tubular structures. Given the rapid and transient nature of MAPK activation in response to stimulation with TNF␣ (27)(28)(29) and the low level of endogenous CD120a (p55) expression, we chose to use a stimulus that resulted in sustained ERK activation, namely okadaic acid, a previously reported TNF␣ mimic (59). Bone marrow-derived mouse macrophages were labeled with [ 32 P]orthophosphate for 6 h, stimulated with 1 M okadaic acid for the last 3 h of labeling, and lysed as described under "Experimental Procedures." CD120a (p55) was immunoprecipitated, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and subjected to autoradiography. As shown in Fig. 11, a low level of basal phosphorylation of CD120a (p55) was detected in unstimulated cells. In contrast, treatment of cells with okadaic acid induced a dramatic increase in the 32 P-labeling of CD120a (p55) and a subsequent gel mobility shift of the phosphorylated receptor. As a control, cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific for the phosphorylated form of ERKs, confirming the activation of ERKs in cells treated with okadaic acid. These findings demonstrate that CD120a (p55) is phosphorylated in intact macrophages following activation of p42 mapk/erk2 .
To determine if activation of p42 mapk/erk2 results in the subcellular redistribution of endogenous CD120a (p55) in intact cells, HeLa cells were treated with okadaic acid for 150 min, fixed, permeabilized, stained for the endogenous CD120a (p55) as described under "Experimental Procedures," and observed by confocal immunofluorescence microscopy. As shown in Fig.  12, untreated cells exhibited a punctate and membranous staining of the receptor similar to that seen in CD120a (p55) transfected cells, although the level of staining was less intense. In contrast, cells treated with okadaic acid showed a redistribution of the receptor to intracellular tubular structures. These tubules were not stained in unpermeabilized cells (data not shown), confirming their intracellular localization. In addition, staining of the receptor in cytoplasmic tubular structures was also observed when staining the endogenous CD120a (p55) in HeLa cells transfected with an expression vector encoding a constitutively active mutant of MEK-1 to drive activation of endogenous p42 mapk/erk2 (Fig. 12). Thus, activation of p42 mapk/erk2 in intact cells leads to the redistribution of the endogenous CD120a (p55) from the plasma membrane to intracellular tubular structures. DISCUSSION Although the ability of cytosolic Ser/Thr protein kinase activities to interact with sequences present in the cytoplasmic domain of CD120a (p55) has been recognized for several years, their identity until now has remained enigmatic. By using both primary cultures of mouse macrophages, and a transfection approach in COS-7 and HeLa cells, the major findings of the work reported herein are that one of these kinases is p42 mapk/erk2 and that the phosphorylation of CD120a (p55) alters the subcellular distribution of the receptor from the plasma membrane and Golgi apparatus to curvilinear tubular structures that by both confocal and electron microscopy appear to be associated with elements of the endoplasmic reticulum. These findings thus provide the following: (i) the first demonstration of inducible phosphorylation of CD120a (p55) in an intact cell system, (ii) defines one of the responsible kinases, and (iii) identifies a novel functional consequence of this event.
Several approaches were used to establish that the inducible GST-CD120a-(207-425) kinase present in mouse macrophages was p42 mapk/erk2 . First, inducible kinase activity co-eluted with p42 mapk/erk2 , but not with other MAPKs including p38 mapk , p46 jnk1 , or p44 mapk/erk1 . Second, pharmacologic antagonism of p42 mapk/erk2 activation with PD098059 or specific immunodepletion of p42 mapk/erk2 substantially inhibited the phosphorylation of GST-CD120a-(207-425) by lysates from stimulated macrophages, whereas depletion or pharmacologic inhibition of other related kinases was without effect. Third, immunoprecipitated catalytically active or recombinant constitutively active p42 mapk/erk2 were able to trans-phosphorylate the GST-CD120a-(207-425) fusion protein in an in vitro kinase assay. These findings therefore suggest that p42 mapk/erk2 , in the absence of other adapter or signaling molecules, is able to interact directly with and phosphorylate sequences present in the cytoplasmic domain of CD120a (p55). Although the site of phosphorylation between residues 207-425 was not fully defined in this study, the cytoplasmic domain does contain four amino acid sequences that conform to the ERK consensus phosphorylation motif PX 1,2 -(S/T)P. Three sites, Thr-236, Ser-240, and Ser-244, are clustered in a Pro-rich region between residues 234 and 245, whereas a fourth site is centered at Ser-270. Significantly, only Ser-244 is conserved within the sequence FSP in all species for which sequence data are available and may therefore represent a candidate site of phosphorylation by p42 mapk/erk2 .
Binding assays using GST-CD120a-(207-425) as an affinity matrix established that p42 mapk/erk2 and p44 mapk/erk1 were capable of physically interacting with the cytoplasmic domain of CD120a (p55). In addition, we have also detected an interaction between p42 mapk/erk2 and the cytoplasmic domain of CD120a (p55) in yeast two-hybrid studies. 2 These findings are also consistent with previously reported studies in other receptor systems. For example, Avery and Dupont (39) have reported that p42 mapk/erk2 co-precipitates with the T-cell receptor com-  2 and 3). The H57 anti-T-cell receptor hamster monoclonal antibody was used as a control for the immunoprecipitation (lane 1). The arrow indicates basal phosphorylation of CD120a (p55); the arrow indicates the gel mobility shift of 32 P-labeled CD120a (p55). As a control, COS cells were transfected with CD120a (p55), and the lysate was separated by SDS-PAGE, transferred onto nitrocellulose, and Western-blotted with antibodies specific for CD120a (p55) (lane 4). B, the cell lysates were also separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with phosphospecific anti-ERK antibodies. plex; and David and colleagues (40) have shown p42 mapk/erk2 to be associated with the interferon ␣/␤-receptor and to participate in IFN␣/␤-dependent signal transduction.
Of the previously described kinase activities capable of phosphorylating residues in the cytoplasmic domain of CD120a (p55), p60 TRAK has been the most thoroughly characterized. p60 TRAK, however, appears distinct from p42 mapk/erk2 based on several criteria. First, substrate specificity experiments using affinity purified p60 TRAK have shown a preference for histone H1 and casein but not for the ERK substrate, myelin basic protein (12). Second, the estimated molecular mass of p60 TRAK of 52 kDa (12) is inconsistent with the molecular mass of p42 mapk/erk2 . Third, p60 TRAK directly or indirectly interacts with residues 344 -397 of human CD120a (p55), a region that is not required for the interaction with p42 mapk/erk2 . 3 Other kinases capable of phosphorylating sequences within the cytoplasmic domain of CD120a (p55) include pp60 src and possibly other as yet to be defined tyrosine kinases that phosphorylate Tyr-331 (41).
Phosphorylation events positively and negatively regulate the functions of many receptors. For example, phosphorylation of Ser and Thr residues in the cytoplasmic domains of the G-protein-coupled formyl-methionyl-leucyl-phenylalanine and CXCR4 receptors has been shown to promote receptor internalization (42,43). In addition, protein kinase C has been shown to phosphorylate the epidermal growth factor receptor at Thr-654, thereby inhibiting the ability of the receptor to stimulate cellular proliferation in the presence of ligand (44). In contrast, phosphorylation of the estrogen receptor by p42 mapk/erk2 has been shown to be required to trans-activate fully estrogenresponsive promoters (45), and the requirement for Tyr phosphorylation of receptors to enable recruitment of SH2-domain containing signaling molecules is now accepted dogma (46,47).
The results of the present study show that phosphorylation of CD120a (p55) by p42 mapk/erk2 induces a striking alteration in the subcellular localization of the receptor in which expression at the plasma membrane and Golgi apparatus is lost, whereas expression within cytoplasmic curvilinear tubular structures is increased. The tubular structures in which the phosphorylated receptor is present were shown to represent distinct elements of the endoplasmic reticulum by three separate methods, namely colocalization with (i) concanavalin A, (ii) VSV-G-KKTN, and (iii) immunolocalization at the electron microscopy level. These findings appeared to be a direct effect of phosphorylation of the receptor itself (as opposed to a change in localization induced by effects of activated p42 mapk/erk2 on other substrates) since when all four ERK consensus sequences in the cytoplasmic domain were mutated to Ala, the redistribution of the receptor was largely prevented. Conversely, when the four ERK consensus sequences were mutated to Asp and Glu to mimic the charge change induced by phosphorylation of Ser and Thr residues, the receptor localized to the curvilinear tubular structures in the absence of activated p42 mapk/erk2 . We also showed that the endogenous CD120a (p55) in mouse macrophages is phosphorylated upon sustained activation of p42 mapk/erk2 and that the endogenous receptor is translocated from the plasma membrane to the curvilinear tubular structures under the same conditions of stimulation in HeLa cells. These findings thus indicate that neither the punctate distribution of the receptor nor the formation of tubular structures are artifacts introduced as a consequence of transient overexpression of CD120a (p55).
Although the spectrum of events affected by the phosphoryl-ation of CD120a (p55) by p42 mapk/erk2 remain to be fully defined, phosphorylation may serve a role in down-regulating receptor function by promoting its translocation from the plasma membrane and Golgi apparatus to the tubular structures. Events analogous to this have been reported for the autocrine motility factor receptor which traffics between plasma membrane caveoli and the endoplasmic reticulum in NIH-3T3 cells (48). Alternatively (or in addition), receptor phosphorylation may prevent newly synthesized CD120a (p55) from trafficking from the endoplasmic reticulum to the Golgi apparatus and subsequently to the plasma membrane. In either situation (i.e. retention in elements of the endoplasmic reticulum or trafficking from the plasma membrane to the endoplasmic reticulum), phosphorylation of the receptor may represent a mechanism to prevent cell surface expression and subsequent signaling functions. This conclusion is intriguing given our current finding that activation of p42 mapk/erk2 by a variety of receptor-ligand interactions results in phosphorylation of GST-CD120a-(207-425). Many receptors undergo homologous and/or heterologous desensitization upon ligand binding or following activation of a panoply of kinases and other signaling molecules (49). In the case of G-protein-coupled receptors including those for ␣and ␤-adrenergic stimuli, glucagon-like peptide-1, and the rhodopsin photoreceptor, desensitization has been shown to be associated with phosphorylation of Ser-and Ser/Thr-rich sequences thereby enabling the binding of ␤-arrestin which subsequently interferes with the ability of the receptor to activate the appropriate heterotrimeric G-protein (50,51). Thus, it is conceivable that activation of p42 mapk/erk2 may promote heterologous desensitization of CD120a (p55) through retrograde translocation of the receptor to cytoplasmic curvilinear tubular structures. An additional and possibly related function of phosphorylation of the cytoplasmic domain of CD120a (p55) by p42 mapk/erk2 may be to recruit additional proteins that promote the accumulation of the receptor within the curvilinear tubular structures. There are few examples of signaling proteins that are capable of interacting with phosphoserine or phosphothreonine although 14-3-3 proteins are a clear example (52). Alternatively, phosphorylation by p42 mapk/erk2 may alter the conformation of the cytoplasmic domain of CD120a (p55) to reveal novel protein interaction motifs. It is also possible that the receptor-associated p42 mapk/erk2 may serve to phosphorylate other proteins that directly or indirectly become recruited to CD120a (p55) and regulate the trafficking of the receptor. It is noteworthy that whereas the death domain region of the receptor has been shown to be required for many downstream functions initiated by ligation of the TNF receptor, the membrane proximal region, which contains the site of phosphorylation by p42 mapk/erk2 , is also required for the expression of nitric oxide synthase (30) although the mechanism of involvement of this region remains unknown.
What happens to the receptor after its accumulation within the curvilinear structures remains unknown. However, the endoplasmic reticulum is known to participate in the degradation of several receptors including the T-cell receptor (53). Recent evidence suggests that these and other receptors probably exit the endoplasmic reticulum through the Sec61c retrograde transporter (54) and undergo degradation in the cytosol in a proteasome-dependent fashion. An additional functional consequence of the phosphorylation of CD120a (p55) may therefore be to promote its ubiquitination. Other studies have also shown that when Bcl-2 is expressed in the endoplasmic reticulum it prevents apoptosis of rat-1/Myc fibroblasts following serum deprivation (55). In addition, expression of Bcl-2 in the endoplasmic reticulum has been shown to assist in the assembly of a putative apoptotic signaling complex containing caspase 8 (56). Intriguingly, filamentous cytoplasmic structures (called death-effector filaments) are formed by transfected FADD, an adapter molecule that is part of the apoptosissignaling complex assembled after engagement of the Fas receptor, as well as by the death-effector domain containing prodomain of procaspase-8 (57). These death-effector filaments recruit procaspase-8, and their formation is associated with induction of apoptosis in Jurkat cells. Thus, perhaps the formation of tubular structures by the phosphorylated TNF receptor CD120a (p55) may also serve a physiologic function distinct from its potential for receptor degradation, such as recruitment of an apoptotic signaling complex.