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Originally published In Press as doi:10.1074/jbc.M312626200 on February 13, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19401-19406, May 7, 2004
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CD81 Associates with 14-3-3 in a Redox-regulated Palmitoylation-dependent Manner*

Krista L. Clark{ddagger}, Alisha Oelke, Megan E. Johnson, Kenneth D. Eilert§, Patrick C. Simpson, and Scott C. Todd

From the Program in Molecular, Cellular and Developmental Biology, Division of Biology, Kansas State University, Manhattan, Kansas 66506

Received for publication, November 19, 2003 , and in revised form, January 25, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N- and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD81 is a member of the tetraspanin family of integral membrane proteins. CD81 was first discovered in 1990 as the target of an antibody with reversible antiproliferative effects on a human B cell lymphoma line (1–4). There are currently 26 membrane proteins classified as members of the tetraspanin family. Members must meet the following criteria: four highly conserved hydrophobic transmembrane domains, two extracellular loops, short intracellular amino and carboxyl tails, and three motifs, CCG, PXSC, and EGC, that contain conserved cysteine residues in the major extracellular domain (5). The smaller extracellular loop is between transmembrane 1 and 2, and the larger extracellular loop, which is likely to mediate interactions with other cell surface proteins, lies between transmembrane 3 and 4.

Many of the initial discoveries of CD81 function were focused in the immune system. On the surface of B cells, CD81 is associated with the CD19·CD21·Leu13 protein complex (6–8). This complex is involved in regulation of B cell receptor signal transduction through recruitment of phosphatidylinositol 3-kinase (PI3K). The CD21·CD19·CD81 complex functions by bringing together required signaling molecules to lower the B cell threshold of activation (9, 10). Similarly, on T cells, CD81 associates with the T cell co-receptors CD4 and CD8 and provides a costimulatory signal with the CD3 subunit of the T cell receptor (11). CD81 may play a role in early T cell development in the thymus (12). The roles for CD81 in immune function were evidenced in 1997 when targeted deletion of CD81 in mice resulted in impaired B-cell proliferation after B-cell antigen-receptor (BCR) cross-linking and hyperproliferation of T cells (13–16). In addition, CD81–/– mice are also less sensitive to allergen-induced airway hyperreactivity, a mouse model of asthma (16).

Another large area of CD81 investigation focuses on its role in pathology. CD81 is required on hepatocytes for human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity (17). Antibodies to CD81 inhibit syncytium formation by human T cell leukemia virus-1 (18, 19). Of great interest, CD81 is a receptor for the E2 envelope protein of hepatitis C virus (HCV).1 Pileri et al. (21) and others (20, 22, 23) showed that the E2 envelope glycoprotein of HCV binds to CD81. Antibodies that neutralize HCV infection in vivo were able to block HCV binding to CD81 in vitro (21). Binding of the viral protein to CD81 may provide the virus with the ability to regulate the immune system. On T cells, CD81 cross-linking is co-stimulatory and has activating effects, whereas on natural killer cells, E2 binding to CD81 is inhibitory to their activation and function (24–26). These results suggest a mechanism whereby HCV binding to CD81 may alter host defenses and enable evasion of the immune response during HCV infection.

Despite the role of CD81 in multiple important cellular functions, the molecular mechanism of action of CD81 in these processes has eluded us. One popular working hypothesis for CD81 function suggests that it is an adapter protein, with the extracellular domain interacting with cell surface proteins and the cytoplasmic tails recruiting intracellular signaling molecules. Potential interactions of the extracellular domain, specifically the large extracellular loop, of CD81 with cell surface molecules are many. CD81 co-immunoprecipitates with a wide variety of cell surface proteins, including members of the integrin family of cell adhesion molecules, growth factors, and a novel member of the Ig superfamily of protein called PGRL (27–30). Which domains in CD81 directly interact with other cell surface proteins remain to be determined. It is known that HCV E2 binds in the helix D region of the large loop of CD81 (31, 32).

Engagement of CD81 by antibody triggers a variety of functional responses. On thymocytes, anti-CD81 generates signals which influence adhesion and proliferation, whereas on mature T cells, CD81 engagement promotes lymphocyte function-associated antigen 1 (LFA-1)-dependent T cell activation, IL-2 production, and proliferation (33). Inhibitors of PKC but not protein tyrosine kinases prevent CD81-induced activation of LFA-1 on T cells. CD81 can also regulate very late antigen (VLA)-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells (34). In B cells, antibodies to CD81 cause a rapid increase in protein tyrosine phosphorylation. Interestingly, tyrosine kinase activity seems to be regulated by the intracellular redox state, but the mechanism of that effect has not been determined (35). Two previous reports (36, 37) demonstrate association of CD81 with PKC and PI3K, but the nature of that interaction is still unknown.

Because tetraspanins have limited intracellular presence, with tails of less than 12 amino acids, their direct role in signaling is frequently overlooked. Signaling through tetraspanin proteins is often attributed to interactions with other membrane proteins. Recent investigations suggest that tetraspanin tails have more functionality than initially thought. The first indication of a functional role for the cytoplasmic tails came in 1996 when Shaw and coworkers (38, 39) showed that CD9 undergoes posttranslational modification by acylation of the amino tail of CD9, though the nature of that acylation was unclear at that time. Attention was again drawn to the intracellular tails when discovery and comparison of six new members of the tetraspanin family revealed conservation of cysteines on the intracellular juxtamembrane portions of the cytoplasmic tails. It was noted that of the 16 tetraspanins known at that time, 15 possessed at least one cysteine in the amino tail, 13 had at least one cysteine in the short intracellular loop between transmembrane 2 and 3, and 14 had cysteines in the carboxyl tail (40). The importance of these cysteines went uninvestigated until recently. While examining another member of the tetraspanin family, CD151, investigators discovered that these conserved cysteines were modified by palmitic acid. Palmitoylation is an acylation modification whereby palmitic acid residues are attached to juxtamembrane intracellular cysteines. Multiple members of the tetraspanin family including CD9, CD81, CD151, and CD63 were examined, and all were modified with palmitic acid (41, 42). Furthermore, a CD151 mutant lacking all intracellular palmitoylation sites showed decreased association with interacting proteins, including CD81, and interfered with proper cellular adhesion and aggregate formation in 293-HEK cells (41, 43). This evidence has relevance for the cytoplasmic tails in CD81 function.

We have employed matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and postsource-decay analysis to identify an intracellular protein that associates with CD81. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. We provide evidence that this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. Furthermore, we find that palmitoylation of CD81 can be regulated by the oxidation state of the cell. These data suggest that 14-3-3 may provide a direct link between CD81 and the range of signaling events resulting from CD81 engagement. The effect of oxidative stress on CD81 palmitoylation and 14-3-3 association may also provide a molecular basis to explain earlier findings that show CD81 signaling to be sensitive to cellular redox (44).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—The human T cell line Jurkat was maintained in RPMI 1640 medium with 10% fetal bovine serum (BioWhittaker, Walkersville, MD). COS-7 and 3T3 were also maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (BioWhittaker). The mouse T cell line MBL was maintained in RPMI 1640 medium with 10% fetal bovine serum (BioWhittaker). Splenocytes were isolated from B6 mice of various ages.

Antibodies—Antibodies used include EAT1 (anti-CD81), 5A6 (anti-CD81), H-8 (anti-14-3-3{beta}) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-14-3-3 (rabbit polyclonal IgG) (Upstate Biotechnology, Lake Placid, NY).

Protein Purification—8 x 108 C6VL cells were lysed in 1% Brij-97. Lysate was run over a column of EAT1 (anti-CD81) antibody covalently crosslinked to protein-A agarose beads (Pierce Endogen, Rockford, IL). Bound protein was eluted in 100 mM glycine, pH 2.8, dialyzed, concentrated, and resolved on 8–16% Tris-glycine gradient gel (Invitrogen). Proteins were visualized with Coomassie Blue, and p30 was excised and subjected to trypsinolysis. MALDI-TOF MS followed by postsource-decay analysis was used to determine the identity of p30 (John Leszyk, University of Massachusetts Medical Center at Worchester, MA).

Palmitoylation—Where indicated, 2 x 107 Jurkat cells were treated with 1 mM H2O2 at 37 °C for 5 min, unless another time is indicated. Diamide (Sigma) was also used at 1 mM for 5 min at 37 °C. Cells were treated overnight with 1 µM L-buthionine-[S,R]-sulfoximine (L-BSO) or 2 mM N-acetyl-L-cysteine (NAC) (Sigma) at 37 °C. After treatment, cells were serum-starved for 2 h in the presence of treating agent. Cells were pelleted and resuspended in 2 ml RPMI 1640 medium + 5% bovine serum albumin. Cells were radiolabeled using palmitic acid [9,10-3H(N)] (PerkinElmer Life Sciences) at 300 µCi/ml for 3 h at 37 °C. Labeled cells were then washed 3x in 5 ml of ice cold PBS, lysed in TBSE buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA) with 1% TX-100 (Sigma) supplemented with 10 units/ml aprotinin (Cal-Biochem, San Diego, CA), 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin A for 1 h on ice and immunoprecipitated as indicated below.

Biotinylation—Two days after transfection, COS-7 cells transiently transfected with indicated CD81 constructs were washed twice in cold PBS and resuspended in 5 ml of PBS supplemented with 2 mM Mg2+ and 2 mM Ca2+. EZ-LinkTM sulfo-NHS-LC Biotin (Pierce Endogen) was added at 1 mg/ml PBS and incubated at room temperature 45 min. Cells were washed 4x in cold PBS and lysed as described for immunoprecipitation.

Immunoprecipitation—Jurkat cells were treated as indicated above or left untreated. Mouse splenocytes were untreated. All cells were lysed at a concentration of 5 x 107 cells/ml in 1 ml of TBSE buffer with 1% Brij-99 (Sigma) supplemented with 10 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin A for 1 h on ice. Confluent 100-mm dishes of transfected COS-7 cells were lysed in the same 1% Brij 99 solution as above also for 1 h at 4 °C. Lysates were clarified by centrifugation for 15 min at 15,000 rpm prior to use. Lysate was precleared for 1 h at 4 °C against 50 µl of goat-anti-mouse (Sigma) or protein-A agarose beads. Anti-14-3-3{beta} and 5A6 were captured on goat-anti-mouse agarose, and EAT1 and 14-3-3{epsilon} at 5 µg of antibody/20 µl of slurry. Antibody was pre-incubated with the indicated beads prior to the addition of 2 x 107 Jurkat or mouse splenocyte cell equivalents, or 500 µl of transfected COS-7 lysate. Samples were rotated overnight at 4 °C. Immunoprecipitated material was washed 4x in lysis buffer, separated on SDS-PAGE, and transferred to PVDF.

Western Blot—Biotinylated material was blotted with streptavidin-HRP (1:20,000) (Southern Biotechnology, Birmingham, AL). 5A6 was used at 1 µg/ml Tris-buffered saline with 1% Tween and 2% bovine serum albumin, anti-14-3-3{beta} antibody was used at 1:100, and both were followed by goat-anti-mouse-HRP (1:20,000) (Southern Biotechnology). Anti-CD81 (EAT1) was used at 1:1000 followed by goat-anti-hamster-HRP (1:40,000) (Southern Biotechnology), and all were visualized using SuperSignalTM West Pico chemiluminescent substrate (Pierce Endogen).

Autoradiography—[3H]Palmitic acid-labeled CD81 immunoprecipitations were also run on SDS-PAGE gel and transferred to PVDF as indicated above. Membranes were dried and exposed on Kodak BioMax MS film using a BioMax Transcreen low energy intensifying screen (Kodak, Rochester, NY) for 1 month at –80 °C.

Cloning and Construction of CD81 and 14-3-3{epsilon} Constructs—Total RNA was isolated from Jurkat cell line using RNeasyTM mini kit (Qiagen, Valencia, CA) and was reverse-transcribed using a reverse transcription system (Promega, Madison, WI). The full-length wild-type CD81 was amplified from this Jurkat cDNA using primers for CD81 (forward, 5'-ATGGGAGTGGAGGGCTGCAC, and reverse, 5'-AGTACACGGAGCTGTTCCGG). The N-terminal CD81 cysteine mutant (NT mut CD81) was made using mutational forward primer 5'-ATGGGAGTGGAGGGTGCCACCAAAGCCATCAAGTACCTG and reverse primer 5'-GTACACGGAGCTGTTCCGG that mutate the two cysteines at amino acids 6 and 9 to alanine. C-terminal CD81 cysteine mutant (CT mut CD81) was generated with wild-type forward primer and mutational reverse primer 5'-GTACACGGAGCTGTTACGGATGCCAGCAGCCAGCACCATGCT that mutate the cysteines at positions 227 and 228 to alanine. The double CD81 cysteine mutant (double mut CD81) was PCR-amplified from Jurkat cDNA using the forward and reverse mutational primers from above, which convert the cysteines in both the N- and C-terminal tails to alanines. Purified PCR products from each of these reactions were cloned into pcDNA3.1/V5His TOPOTM TA (Invitrogen). The triple CD81 cysteine mutant (triple mut CD81) lacking all intracellular cysteines was generated with QuikChangeTM site-directed mutagenesis kit (Stratagene, Cedar Creek, TX), according to the manufacturer's specifications. The CD81 double cysteine mutant in pcDNA3.1/V5His TOPOTM TA was used as a template, with overlapping mutant primer 5'-CATCCAGGAATCCCAGTCCCTGCTGGGGACGTTC and reverse primer 5'-GAACGTCCCCAGCAGGGACTGGGATTCCTGGATG. This final mutagenesis converts the cysteine at position 89 in the intracellular loop between transmembranes 2 and 3 to alanine. All constructs were verified by sequencing. Although the pcDNA3.1/V5His TOPOTM TA vector does provide for an epitope tag, the V5-His tag is not transcribed because of the endogenous CD81 stop codon.

Transfection—24 hours prior to transfection, COS-7 cells were trypsinized and plated at 70–80% confluency in a 100-mm tissue culture dish. LipofectAMINE 2000 (Invitrogen) was used for transfections according to the manufacturer's protocol. COS-7 was transfected with 20 µg of the indicated CD81 mutant plasmid. Cells were cultured 48 h at 37 °C prior to lysis in 1 ml of 1% Brij-99 lysis solution. Immunoprecipitation, radiolabeling, and Western blotting were carried out as stated above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD81 Association with 14-3-3{epsilon}—The next step in understanding the mechanism of CD81 function is elucidating the signal transduction pathway utilized by CD81. In our previous investigation, we identified surface proteins associated with CD81 through biotinylation patterns of CD81 immunoprecipitations. This method does not allow for the identification of intracellular CD81-associated proteins. We began looking for CD81-associated intracellular proteins by analyzing proteins visualized by Coomassie Blue but which are not labeled by biotinylation of the cell surface. CD81-associated proteins were isolated from 1% Brij-97 lysate of the mouse T cell line MBL. The lysate was run over a column of anti-CD81 antibody (EAT1) antibody and were eluted with 100 mM glycine, pH 2.8. The eluate was dialyzed, concentrated, and resolved on 8–16% Tris-glycine gradient gel. The associated proteins were visualized with Coomassie Blue, and proteins that were unique to the Coomassie-stained gel, yet not seen by Western blotting with avidin-HRP, were targeted for identification (Fig. 1A). To preserve the majority of tetraspanin interactions, it was necessary to use a less stringent detergent, such as Brij-97. Because of the highly interactive nature of tetraspanin proteins, these complexes often contain multiple proteins. These protein complexes do have a discrete size and have been shown to have functional relevance. From the Coomassie-stained gel of the anti-CD81 immunoprecipitation shown in Fig. 1A, we selected the most prominent protein at ~30 kDa for identification. p30 was excised and subjected to trypsinolysis. MALDI-TOF MS followed by postsource-decay analysis was used to determine the identity of p30. The six peptide sequences derived from fragments of p30, KDSTLIMQLLR, RYLAEFATGNDRK, KVAGMDVELTVEERN, MDDREDLVYQKL, KAASDIAMTELPPTHPIRL, and KLAEQAERYDEMVESMKK, are shown in table format in Fig. 1B. Each of these peptide sequences is a 100% match for 14-3-3{epsilon}, and together these peptides cover 34.5% (88/255 amino acids) of the whole 14-3-3{epsilon} protein. These peptide fragments have been highlighted within the complete protein sequence of 14-3-3{epsilon} shown in Fig. 1C. The consensus binding sequence of 14-3-3 is a phosphoserine (pS) flanked by an arginine, e.g. RSXpSXP and RX(Y/F)XpSXP or RXSX(S/T)XP. CD81 has a sequence of RNSSVY in its amino-terminal tail, which bears a striking similarity to the 14-3-3 consensus sequence. The terminal proline in known 14-3-3 binding sites aligns with the carboxyl terminus of CD81, and if the role of the proline is to bend subsequent polypeptide away from the docking site, then this may not be necessary at the terminus of CD81.



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  		FIG. 1.
p30 MALDI-TOF MS peptide fragments. A, Coomassie Blue stain of the anti-CD81 immunoprecipitation in MBL mouse T cells. B, MALDI-TOF MS of p30 yields six peptide fragments, shown here with indicated MH+ values and the results of postsource-decay, if completed. Peptide fragments are underlined in black in the context of the complete amino acid sequence of 14-3-3{epsilon}. C, double-underlined fragment indicates overlap of two individual fragments. Each of these peptide fragments is a 100% match for 14-3-3{epsilon} and, combined, the fragments cover 34.5% of the total protein. D, physiologic association of CD81 with 14-3-3 was verified by co-immunoprecipitation in primary mouse splenocytes.

 
To confirm the physiologic association of 14-3-3{epsilon} with CD81, we conducted co-immunoprecipitation experiments in primary mouse splenocytes. As seen in Fig. 1D, CD81 does indeed coprecipitate with 14-3-3 from Brij-99 lysate of primary mouse splenocytes.

Regulation of CD81 Palmitoylation—We also examined the association of CD81 with 14-3-3{epsilon} in human cell lines. For these experiments, we used an antibody to 14-3-3 that recognizes a structural region common to all seven isoforms. Our initial attempts to co-immunoprecipitate these two proteins from human cell lines were unsuccessful. We theorized that the association of CD81 with 14-3-3 might be constitutive in mouse but inducible in human cell lines. As we considered events that might regulate the interaction of 14-3-3 with the tail of CD81, two papers were published showing that multiple members of the tetraspanin family, including CD9, CD81, CD151 and CD63, are posttranslationally modified with palmitic acid on the juxtamembrane cysteines of their intracellular tails (41, 42).

The tails of CD81 are short, with only 11 amino acids in the N-terminal tail and 12 amino acids in the C-terminal tails. CD81 has two cysteines on its N-terminal tail, two in its C-terminal tail, and one on the intracellular loop between its two large extracellular loops. These cysteines provide a potential for the covalent linking of up to five palmitic acids. We hypothesize that the presence of lipid moieties on these relatively short intracellular tails may block access of 14-3-3 to its potential binding site within those same tails. If a cellular mechanism for regulation of the presence of these palmitic acid residues exists, it would in turn provide a method of controlling 14-3-3 access to the potential binding domain on the intracellular tail of CD81. With this idea in mind, we began to search for cellular treatments that might manipulate the palmitoylation state of CD81.

A study examining the effects of cellular redox on endothelial cells demonstrates that exposure of endothelial cells to oxidative stress markedly inhibits the palmitoylation of caveolin-1 (45). Based on this evidence, we investigated the effects of cellular redox manipulation on the association of CD81 with 14-3-3. Glutathione (GSH) is a tripeptide thiol compound found in many prokaryotes and eukaryotes that serves as an intracellular buffering system and protects cells from damages by intracellular free radicals. Here we use three oxidative stress inducers: L-BSO, which depletes glutathione levels by inactivating {gamma}-glutamyl-cysteine synthetase, one of the enzymes responsible for production of GSH; diamide, which is a glutathione-oxidizing agent; and H202, which increases the presence of free radicals requiring higher levels of GSH to buffer, thus resulting in depleted levels of GSH. NAC is a cysteine analogue that increases GSH levels and serves as an antioxidant. Jurkat cells were serum-starved for 2 h, resuspended in 2 ml of RPMI 1640 medium, 5% bovine serum albumin and were left untreated or treated with H2O2, diamide, L-BSO, or NAC, as indicated under "Materials and Methods." Incorporation of palmitic acid was then evaluated as described (41, 43). Cells were lysed in 1% TX-100, and CD81 was immunoprecipitated from each of the lysates using 5A6 (anti-CD81). Samples were washed, eluted, and separated on 8–16% gradient SDS-PAGE gels, and transferred to PVDF. Membranes were dried and exposed to film for 1 month at –80 °C.

CD81 palmitoylation is significantly decreased in the presence of the oxidative stress inducing reagents L-BSO, diamide, and H202, as shown in Fig. 2. In fact, in the presence of H202, palmitoylation appears to be completely inhibited. In contrast, in the presence of the antioxidant NAC, CD81 palmitoylation remains at levels similar to untreated cells.



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  		FIG. 2.
Palmitoylation of CD81 regulated by oxidative stress. 2 x 107 Jurkat cells per sample were treated as follows: 1 mM H2O2 at 37 °C for 5 min; 1 mM diamide for 5 min at 37 °C; 1 µM L-BSO at 37 °C overnight; 2 mM NAC at 37 °C overnight. After treatment, cells were radiolabeled using [3H]palmitic acid at 300 µCi/ml and lysed in TX-100. Samples were immunoprecipitated with 5A6, separated on SDS-PAGE, and transferred to PVDF. Membranes were dried and exposed on Kodak BioMax MS film using a BioMax Transcreen low energy intensifying screen (Kodak) for 1 month at –80 °C. Subsequently, the membrane was Western blotted with 5A6 to verify presence of CD81.

 
H202 Regulates CD81 Association with 14-3-3—After observing that oxidative stress induced by H202 does inhibit palmitoylation of CD81, we began experiments to test the second part of our hypothesis: does H202-mediated inhibition of CD81 palmitoylation affect its association with 14-3-3? To test this hypothesis, 2 x 107 Jurkat cells per sample were treated with 1 mM H202 for 5 min at 37 °C. After treatment, cells were lysed, and lysate of the samples was divided in half. One sample was immunoprecipitated with 5A6, and the other with anti-14-3-3. Precipitated proteins were separated on SDS-PAGE and transferred. As seen in Fig. 3A, 14-3-3 immunoprecipitation in Jurkat cells in the absence of H2O2 shows no co-immunoprecipitation with CD81. But upon 5 min of stimulation with H202, CD81 is readily visible in a 14-3-3 immunoprecipitation. A time course of activation shown in Fig. 3B indicates that the association of CD81 with 14-3-3 becomes identifiable by Western blotting after only 10 s and continues to increase up to 30 s.



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  		FIG. 3.
H202-induced association of CD81 with 14-3-3. Samples containing 2 x 107 Jurkat cells each were left untreated or treated with 1 mM H2O2 at 37 °C for 5 min (A) or the indicated time period (B) and then lysed in 1% Brij-99. Each lysate sample was divided, and half was immunoprecipitated with 5A6 and the other with anti-14-3-3. Samples were separated on SDS-PAGE, transferred to PVDF, and Western blotted with 5A6 (A) or anti-14-3-3 (B).

 
CD81 Palmitoylation-defective Mutant Constitutively Associates with 14-3-3—If the mechanism of H202 regulation of CD81 association with 14-3-3 is through inhibition of palmitoylation of CD81, then a CD81 molecule that could not be palmitoylated may be constitutively associated with 14-3-3. To test this hypothesis, we generated a series of CD81 cysteine mutants. There are a total of five cysteines in the intracellular portion of CD81. On the N-terminal tail, there are cysteines at amino acids 6 and 9. On the internal loop between TM2 and TM3, there is a cysteine at amino acid 89, and on the C-terminal tail there are two additional cysteines at amino acids 227 and 228. We generated a series of five CD81 constructs: wild-type CD81; CD81 with mutation in both the cysteines of the N-terminal tail (NT mut CD81); CD81 with mutation in both the cysteines of the C-terminal tail (CT mut CD81); CD81 with mutation in the cysteines of both the N and C-terminal tail (double mut CD81); and CD81 with mutations at all five of the intracellular cysteines (triple mut CD81). All of these constructs are expressed in the pcDNA3.1/V5His TOPOTM TA vector. We utilized the native CD81 stop codon to prevent translation of the epitope tag. To verify that each of these constructs was expressed, COS-7 cells were transiently transfected with each vector, cultured 2 days, and lysed prior to immunoprecipitation with 5A6 followed by anti-CD81 Western blotting. Each vector produced proteins of the expected sizes and in similar amounts, and all constructs were expressed at the cell surface, as determined by surface biotinylation followed by streptavidin Western blotting (Fig. 4A).



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  		FIG. 4.
CD81 lacking palmitoylation is constitutively associated with 14-3-3. COS-7 was transfected with the indicated CD81 plasmid and with control cells left untransfected. After 48 h, cells were lysed in 1% Brij-99 lysis solution. Biotinylated samples were lysed in TX-100. A, all lysate was immunoprecipitated with 5A6, separated on SDS-PAGE, and Western blotted with either 5A6 or streptavidin-HRP, as indicated. To determine palmitoylation state of the CD81 mutants 48 h after transfection, cells were radiolabeled using [3H]palmitic acid at 300 µCi/ml and lysed in TX-100. Samples were immunoprecipitated with 5A6, separated on SDS-PAGE, and transferred to PVDF. Membranes were dried and exposed on Kodak BioMax MS film using a BioMax Transcreen low energy intensifying screen (Kodak) for 1 month at –80 °C. B, subsequently, the membrane was Western blotted with 5A6 to verify presence of CD81. C, the same 5A6 immunoprecipitations of CD81 mutant-transfected COS-7 cells from 4A were separated on SDS-PAGE and blotted with anti-14-3-3.

 
To verify the palmitoylation state of each of the mutants, transiently transfected COS-7 cells were transfected with each of the CD81 plasmids, and 2 days after transfection, cells were radiolabeled with [3H]palmitic acid as indicated above. Mutation in the N- or C-terminal tails individually does not seem to reduce the overall level of palmitic acid incorporated into the CD81 molecules. The double mutant shows a slight reduction in palmitic acid incorporation, suggesting palmitoylation of the small intracellular loop between TM2 and TM3. This speculation is confirmed by mutation of the final cysteine of the intracellular loop, as the triple mutant shows a significant reduction in the level of palmitic acid incorporation (Fig. 4B).

If CD81 association with 14-3-3 requires CD81 to be unpalmitoylated, only the CD81 triple mutant would be expected to associate with 14-3-3 constitutively. To test this, the same 5A6 (anti-CD81) immunoprecipitations of the CD81 mutant-transfected COS cells shown in Fig. 4A were run on a second SDS-PAGE gel, transferred, and Western-blotted with the 14-3-3 antibody. This experiment tested for association of the recombinant CD81 constructs with endogenous 14-3-3. It should be noted here that the intracellular tails of monkey CD81 are identical to that of the human counterpart. Recall that this antibody is directed against a conserved epitope and recognizes all seven isoforms of 14-3-3. As shown in Fig. 4C, only the triple mutant of CD81, which lacks all five palmitoylation sites, was found constitutively associated with 14-3-3. Because of the cross-reactivity of the antibody, we were not able to identify the specific 14-3-3 isoform associated with the triple cysteine mutant of CD81, but based on the MALDI-TOF MS data, the epsilon isoform is a strong candidate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The 14-3-3 proteins are a highly conserved family of dimeric proteins. There are seven known isoforms of 14-3-3 in mammals. This family of proteins regulates enzymes, controls subcellular localization of associated proteins, and functions as adaptor proteins. The consensus binding motif of 14-3-3 is R(S/Ar)XpSXP and RX(Ar/S)XpSXP, although multiple binding motifs have been identified. Although phosphorylation was initially believed to be a requirement for binding to 14-3-3, some isoforms do not require phosphorylation (46). CD81 has a sequence of RNSSVY in its amino-terminal tail, which bears a striking similarity to the 14-3-3 consensus sequence, thus providing a structural basis for the association. A link between CD81 and 14-3-3 may provide a mechanistic explanation. For example, 14-3-3 may mediate the recruitment of PKC and/or PI3K to CD81, as described previously (36, 37).

During normal and pathological conditions, cells experience oxidative stress caused by multiple environmental factors such as inflammatory by-products, oxygen deprivation, or GSH depletion. According to the data presented in Fig. 2, such oxidative stress inhibits palmitoylation of CD81. In the absence of the palmitic acid residues, the putative 14-3-3 binding motif could be exposed, allowing for its association with the CD81 tail. This possibility suggests that CD81-mediated signaling may differ under normal versus oxidative stress conditions. This may explain previously observed differences in tyrosine signaling in redox environments (44).

14-3-3 proteins regulate the function of many isoforms of PKC as well as PI3K (reviewed in Ref. 46). Co-immunoprecipitation and covalent cross-linking experiments show that the tetraspanins CD9, CD53, CD81, CD82, and CD151 associate with activated PKC and link PKC to specific {beta}1 integrins (36). A recent paper by Feigelson et al. (34) suggests that CD81 regulation of VLA-4 ({alpha}4{beta}1) is a PKC-triggered effect upon avidity. In addition, PKC inhibitors interfere with CD81-induced activation of LFA-1 (47). There are also reports of co-immunoprecipitation of {alpha}3{beta}1, CD81, and PI4K (37). A controlled association between CD81 and 14-3-3 could provide a mechanism for CD81 regulation of PKC activity, which may in turn allow for modulation of integrin function.

In conclusion, we have identified the signal transduction molecule 14-3-3{epsilon} in association with the tetraspanin protein CD81. In human cell lines, the association of CD81 with 14-3-3 is regulated by the palmitoylation state of the CD81 intracellular tails. Upon induction of cellular oxidation, incorporation of palmitic acid residues into CD81 is inhibited. In the unpalmitoylated state, CD81 association with 14-3-3 is induced. Future studies are needed to see if CD81 can associate with multiple 14-3-3 isoforms or if this association is specific to the epsilon isoform. At least eight other tetraspanins contain a similar intracellular tail sequence, with an arginine followed closely by a serine, suggesting other tetraspanins may also be capable of 14-3-3 binding. At this time, it is not known if other tetraspanins can also associate with 14-3-3. CD81 association with 14-3-3 may represent an important link between CD81 extracellular interactions and its ability to trigger functional signal transduction.


   FOOTNOTES
 
* This research was supported by National Institutes of Health Grant AI052206 [GenBank] . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by National Institutes of Health Grant RR16475. Back

{ddagger} To whom correspondence should be addressed: 239C Chalmers Hall, Division of Biology, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-6795; Fax: 785-532-6653; E-mail: c6vl{at}hotmail.com.

1 The abbreviations used are: HCV, hepatitis C virus; pS, phosphoserine; PI3K, phosphatidylinositol 3-kinase; IL, interleukin; LFA-1, lymphocyte function-associated antigen 1; VLA, very late antigen; L-BSO, L-buthionine-[S,R]-sulfoximine; NAC, N-acetyl-L-cysteine; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; PKC, protein kinase C. Back


   ACKNOWLEDGMENTS
 
We thank Drs. S. K. Chapes, R. J. Clem, and A. L. Passarelli for their continued support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wright, M. D., Henkle, K. J., and Mitchell, G. F. (1990) J. Immunol. 144, 3195–3200[Abstract]
  2. Oren, R., Takahashi, S., Doss, C., Levy, R., and Levy, S. (1990) Mol. Cell. Biol. 10, 4007–4015[Abstract/Free Full Text]
  3. Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955–2962[Abstract/Free Full Text]
  4. Classon, B. J., Williams, A. F., Willis, A. C., Seed, B., and Stamenkovic, I. (1990) J. Exp. Med. 172, 1007[Free Full Text]
  5. Hemler, M. E. (2001) J. Cell Biol. 155, 1103–1107[Abstract/Free Full Text]
  6. Bradbury, L. E., Kansas, G. S., Levy, S., Evans, R. L., and Tedder, T. F. (1992) J. Immunol. 149, 2841–2850[Abstract]
  7. Takahashi, K., Nakamura, T., Koyanagi, M., Kato, K., Hashimoto, Y., Yagita, H., and Okumura, K. (1990) J. Immunol. 145, 4371–4379[Abstract]
  8. Fearon, D. T., and Locksley, R. M. (1996) Science 272, 50–53[Abstract]
  9. Matsumoto, A. K., Martin, D. R., Carter, R. H., Klickstein, L. B., Ahearn, J. M., and Fearon, D. T. (1993) J. Exp. Med. 178, 1407–1417[Abstract/Free Full Text]
  10. Tedder, T. F., Zhou, L. J., and Engel, P. (1994) Immunol. Today 15, 437–442[CrossRef][Medline] [Order article via Infotrieve]
  11. Levy, S., Todd, S. C., and Maecker, H. T. (1998) Annu. Rev. Immunol. 16, 89–109[CrossRef][Medline] [Order article via Infotrieve]
  12. Boismenu, R., Rhein, M., Fischer, W. H., and Havran, W. L. (1996) Science 271, 198–200[Abstract]
  13. Maecker, H. T., and Levy, S. (1997) J. Exp. Med. 185, 1505–1510[Abstract/Free Full Text]
  14. Miyazaki, T., Muller, U., and Campbell, K. S. (1997) EMBO J. 16, 4217–4225[CrossRef][Medline] [Order article via Infotrieve]
  15. Tsitsikov, E. N., Gutierrez-Ramos, J. C., and Geha, R. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10844–10849[Abstract/Free Full Text]
  16. Deng, J., Yeung, V. P., Tsitoura, D., DeKruyff, R. H., Umetsu, D. T., and Levy, S. (2000) J. Immunol. 165, 5054–5061[Abstract/Free Full Text]
  17. Silvie, O., Rubinstein, E., Franetich, J. F., Prenant, M., Belnoue, E., Renia, L., Hannoun, L., Eling, W., Levy, S., Boucheix, C., and Mazier, D. (2003) Nat. Med. 9, 93–96[CrossRef][Medline] [Order article via Infotrieve]
  18. Imai, T., Fukudome, K., Takagi, S., Nagira, M., Furuse, M., Fukuhara, N., Nishimura, M., Hinuma, Y., and Yoshie, O. (1992) J. Immunol. 149, 2879–2886[Abstract]
  19. Imai, T., and Yoshie, O. (1993) J. Immunol. 151, 6470–6481[Abstract]
  20. Petracca, R., Falugi, F., Galli, G., Norais, N., Rosa, D., Campagnoli, S., Burgio, V., Di Stasio, E., Giardina, B., Houghton, M., Abrignani, S., and Grandi, G. (2000) J. Virol. 74, 4824–4830[Abstract/Free Full Text]
  21. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, S. (1998) Science 282, 938–941[Abstract/Free Full Text]
  22. Flint, M., Maidens, C., Loomis-Price, L. D., Shotton, C., Dubuisson, J., Monk, P., Higginbottom, A., Levy, S., and McKeating, J. A. (1999) J. Virol. 73, 6235–6244[Abstract/Free Full Text]
  23. Higginbottom, A., Quinn, E. R., Kuo, C. C., Flint, M., Wilson, L. H., Bianchi, E., Nicosia, A., Monk, P. N., McKeating, J. A., and Levy, S. (2000) J. Virol. 74, 3642–3649[Abstract/Free Full Text]
  24. Wack, A., Soldaini, E., Tseng, C., Nuti, S., Klimpel, G., and Abrignani, S. (2001) Eur. J. Immunol. 31, 166–175[CrossRef][Medline] [Order article via Infotrieve]
  25. Tseng, C. T., and Klimpel, G. R. (2002) J. Exp. Med. 195, 43–49[Medline] [Order article via Infotrieve]
  26. Crotta, S., Stilla, A., Wack, A., D'Andrea, A., Nuti, S., D'Oro, U., Mosca, M., Filliponi, F., Brunetto, R. M., Bonino, F., Abrignani, S., and Valiante, N. M. (2002) J. Exp. Med. 195, 35–41[Medline] [Order article via Infotrieve]
  27. Boucheix, C., and Rubinstein, E. (2001) Cell Mol. Life Sci. 58, 1189–1205[CrossRef][Medline] [Order article via Infotrieve]
  28. Lagaudriere-Gesbert, C., Le Naour, F., Lebel-Binay, S., Billard, M., Lemichez, E., Boquet, P., Boucheix, C., Conjeaud, H., and Rubinstein, E. (1997) Cell. Immunol. 182, 105–112[CrossRef][Medline] [Order article via Infotrieve]
  29. Clark, K. L., Zeng, Z., Langford, A. L., Bowen, S. M., and Todd, S. C. (2001) J. Immunol. 167, 5115–5121[Abstract/Free Full Text]
  30. Stipp, C. S., Kolesnikova, T. V., and Hemler, M. E. (2001) J. Biol. Chem. 276, 40545–40554[Abstract/Free Full Text]
  31. Drummer, H. E., Wilson, K. A., and Poumbourios, P. (2002) J. Virol. 76, 11143–11147[Abstract/Free Full Text]
  32. VanCompernolle, S. E., Wiznycia, A. V., Rush, J. R., Dhanasekaran, M., Baures, P. W., and Todd, S. C. (2003) Virology 314, 371–380[CrossRef][Medline] [Order article via Infotrieve]
  33. VanCompernolle, S. E., Levy, S., and Todd, S. C. (2001) Eur. J. Immunol. 31, 823–831[CrossRef][Medline] [Order article via Infotrieve]
  34. Feigelson, S. W., Grabovsky, V., Shamri, R., Levy, S., and Alon, R. (2003) J. Biol. Chem. 278, 51203–51212[Abstract/Free Full Text]
  35. Schick, M. R., and Levy, S. (1993) J. Immunol. 151, 4090–4097[Abstract]
  36. Zhang, X. A., Bontrager, A. L., and Hemler, M. E. (2001) J. Biol. Chem. 276, 25005–25013[Abstract/Free Full Text]
  37. Berditchevski, F., Tolias, K. F., Wong, K., Carpenter, C. L., and Hemler, M. E. (1997) J. Biol. Chem. 272, 2595–2598[Abstract/Free Full Text]
  38. Seehafer, J. G., Slupsky, J. R., Tang, S. C., and Shaw, A. R. (1988) Biochim. Biophys. Acta 952, 92–100[CrossRef][Medline] [Order article via Infotrieve]
  39. Seehafer, J. G., Tang, S. C., Slupsky, J. R., and Shaw, A. R. (1988) Biochim. Biophys. Acta 957, 399–410[CrossRef][Medline] [Order article via Infotrieve]
  40. Todd, S. C., Doctor, V. S., and Levy, S. (1998) Biochim. Biophys. Acta 1399, 101–104[Medline] [Order article via Infotrieve]
  41. Yang, X., Claas, C., Kraeft, S. K., Chen, L. B., Wang, Z., Kreidberg, J. A., and Hemler, M. E. (2002) Mol. Biol. Cell 13, 767–781[Abstract/Free Full Text]
  42. Berditchevski, F., Gilbert, E., Griffiths, M. R., Fitter, S., Ashman, L., and Jenner, S. J. (2001) J. Biol. Chem. 276, 41165–41174[Abstract/Free Full Text]
  43. Berditchevski, F., Odintsova, E., Sawada, S., and Gilbert, E. (2002) J. Biol. Chem. 277, 36991–37000[Abstract/Free Full Text]
  44. Schick, M. R., Nguyen, V. Q., and Levy, S. (1993) J. Immunol. 151, 1918–1925[Abstract]
  45. Parat, M. O., Stachowicz, R. Z., and Fox, P. L. (2002) Biochem. J. 361, 681–688[CrossRef][Medline] [Order article via Infotrieve]
  46. van Hemert, M. J., Steensma, H. Y., and van Heusden, G. P. (2001) BioEssays 23, 936–946[CrossRef][Medline] [Order article via Infotrieve]
  47. Todd, S. C., Lipps, S. G., Crisa, L., Salomon, D. R., and Tsoukas, C. D. (1996) J. Exp. Med. 184, 2055–2060[Abstract/Free Full Text]

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