The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jin, Y.-J.
Right arrow Articles by Burakoff, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jin, Y.-J.
Right arrow Articles by Burakoff, S. J.
The Journal of Immunology, 2004, 173: 5495-5500.
Copyright © 2004 by The American Association of Immunologists

CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD41

Yong-Jiu Jin2,*, Xiaoping Zhang{ddagger}, J. Gildade Boursiquot* and Steven J. Burakoff*,{dagger}

* Skirball Institute of Biomedical Research, {dagger} Department of Medicine, New York University School of Medicine, New York, NY 10016; and {ddagger} Department of Pharmaceutics, College of Pharmacy, Rutgers University, Piscataway, NJ 08854


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (~95%), whereas that induced by PMA was partial (~40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 is a coreceptor of the TCR in TCR-mediated T cell activation and signaling. Cell surface CD4 can be down-regulated by PMA treatment (1, 2). PMA treatment activates protein kinase C (PKC),3 which, in turn, induces the phosphorylation of Ser408 and Ser415 in the cytoplasmic tail of CD4 (3, 4). As a result, CD4 dissociates from Lck and is recruited into the clathrin-coated pits for endocytosis (5, 6). After endocytosis, some of the internalized CD4 molecules are delivered to lysozomes for degradation, whereas others recycle back to the T cell surface (1, 5). Therefore, PMA-induced down-regulation of CD4 is incomplete. In T cells, which express Lck, PMA treatment removes 40–50% of CD4 molecules from the cell surface; in CD4-transfected HeLa cells, which do not express Lck, PMA removes 75% of the surface-expressed CD4 molecules (4, 6).

CD4 is also the primary receptor for HIV to enter cells. HIV Nef protein, expressed early in viral replication (7), is a major determinant of HIV virulence (8, 9, 10, 11). One well-characterized Nef function is the down-regulaton of CD4 from the cell surface (12), which correlates with Nef-dependent enhancement of viral pathogenicity (13, 14, 15, 16, 17, 18, 19, 20).

The Nef down-regulation of CD4 is independent of the phosphorylation of CD4 (12). The available data indicate that Nef down-regulation of CD4 requires its interaction with CD4 on the T cell plasma membrane (21, 22, 23, 24). The CD4 tail is necessary for Nef down-regulation of CD4 (21, 22), as is the membrane association of Nef through its N-terminal myristoylation (25). In Nef, W57 and L58 are the residues critical for the Nef-CD4 interaction (26, 27). Replacing the CD4 tail with Nef results in a chimeric protein that down-regulates CD4 in trans as well as the chimeric protein itself in cis (23). Several studies have shown that CD4 interacts directly with Nef in vitro and in insect cells and that the dileucine motif in the CD4 tail is essential for the interaction (25, 26, 28, 29). However, the direct interaction between CD4 and Nef in mammalian cells has yet to be demonstrated (24).

Available data also indicate that Nef links CD4 to the clathrin-coated pits for endocytosis, but how Nef does this remains controversial. Nef contains a highly conserved dileucine motif (L164-L165) in its C-terminal flexible loop, which is essential for Nef down-regulation of CD4 (30, 31, 32). The dileucine motif, including the one in the CD4 tail (2), has the consensus sequence of [DE]XXXL[LI]. The dileucine motifs are known sorting signals, recognizable by adaptor protein complexes, including AP-1, AP-2, AP-3, and AP-4 (33). The prevailing view is that the adaptor protein-2 (AP-2) is involved in endocytosis from plasma membranes, whereas AP-1 and AP-3 are involved in the trans-Golgi network and lysosome vesicular trafficking, respectively (34). It has been shown that the colocalization of Nef with the AP-2 complex is correlated with Nef down-regulation of CD4 (35). However, yeast two- or three-hybrid studies show that the dileucine motif in HIV Nef interacts mainly with AP-1 and AP-3 and only weakly with AP-2 (36, 37). A GST-tagged HIV Nef binds to AP-1, but not AP-2 (38). Apparently, CD4-Nef interacts with an AP complex, but the specific AP complex involved needs to be further defined.

In an effort to elucidate the differential pathways of CD4 internalization induced by Nef and PMA, we found that Nef down-regulation of CD4 was partially reversed in Jurkat T cells by treatment of PMA. Further analysis suggested that phosphorylation of CD4 induced by PMA disrupted the interaction between Nef and the CD4 tail. A model is proposed in which the Nef down-regulation of CD4 is mediated through a CD4-Nef-AP interaction, whereas the PMA down-regulation of CD4 is through a direct interaction of the phosphorylated CD4 with an AP complex. PMA switches the connection of CD4 from the former route to the later, resulting in a net gain of CD4 on the T cell surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Abs, and chemicals

The Jurkat T cell line J77, a variant of clone E6-1 (from American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium containing 10% FBS at 37°C in a 5% CO2 humidified atmosphere. PE-conjugated anti-CD4 (Leu 3A) was purchased from BD Immunocytometry (San Diego, CA), and polyclonal anti-CD4 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). mAb OKT4D was prepared from a hybridoma purchased from American Type Culture Collection. Anti-HIV-1 Nef rabbit serum was obtained from the National Institutes of Health AIDS Research and Reference Reagent Repository provided by Dr. R. Swanstrom. The PKC inhibitor bisindolylmaleimide1 (BIM1) was purchased from Calbiochem (San Diego, CA), and 3,3-dithiobis[sulfosuccinimidyl-propionate] (DTSSP) was obtained from Pierce (Rockford, IL).

Generation of mutant CD4 and HIV Nef

The construction and the stable expression of wild-type (wt) CD4, 2C CD4, and tail-less CD4 in Jurkat T cells have been described previously (39). The serine mutant of CD4 used in this study was similarly constructed. The PCR product containing Ser to Ala substitutions at residues 408 and 415 was digested with SfoI/BamHI, and the resultant fragment was used to replace the corresponding wt CD4 fragment in wt CD4 cDNA originally cloned in pcDNA3 vector (Invitrogen Life Technologies, Carlsbad, CA). The CD4 Ser mutant plasmid was then stably transfected into Jurkat T cells. Nef (NA7)-GFP plasmid was provided by Dr. J. Skowronski (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY: Ref. 35). The non-GFP-tagged Nef was constructed by PCR subcloning of Nef (NA7) into pcDNA3 vector (Invitrogen Life Technologies). NefLL, NefWL, NefLL-GFP, and NefWL-GFP were generated by PCR mutagenesis using Nef or Nef-GFP as the template following the protocol of the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). All mutations generated in this study were confirmed by DNA sequencing.

PMA treatment and two-color cytometric analysis

For transient expression, 20 µg of double CsCl gradient-purified plasmid DNA was electroporated into 5 x 106 CD4 T cells at 800 µF and 250 V. Sixteen to 18 h after transfection, cells were treated with 100 ng/ml PMA in RPMI 1640 medium containing 1 mg/ml BSA at 37°C for 15 min. For CD4 staining, cells were incubated with PE-conjugated anti-CD4 Ab diluted 1/100 in PBS on ice for 45 min. Cells were then subjected to FACS using a FACScan (BD Biosciences, Mountain View, CA). In FACS analysis, vertical and horizontal lines were drawn to demarcate CD4- and Nef-GFP-positive and -negative cell populations. The line settings were based on the cell surface staining intensities of CD4 and GFP, which varied somewhat among transfected cell lines. The relative cell surface CD4 expression (percentage) was the ratio of the medium CD4 staining of PMA-treated cells (+PMA) to that of PMA-untreated cells (–PMA). The 100% level (steady state) was the CD4 expression in mock-transfected control cells without PMA treatment. Each value was the average of three independent experiments (mean ± SD).

Subcellular fractionation, chemical cross-linking, and immunoblotting

The subcellular fractionation of Jurkat cells and the immunoblotting were conducted as previously described (39). After washing with PBS, cells were incubated in hypotonic buffer (10 mM Tris (pH 7.5) and 330 mM sucrose) for 10 min on ice. Cells were then passed 10 times through a 30-gauge needle while on ice. The extract was centrifuged at 250 x g for 10 min to remove the nuclei and intact cells. To separate the cytosolic fraction from membrane, the postnuclear supernatant was centrifuged at 50,000 rpm for 1 h using a TL-100 centrifuge and a TLS-55 rotor (Beckman Coulter, Fullerton, CA).

For chemical cross-linking, the membrane fraction from 107 T cells was resuspended in 100 µl of PBS, to which 100 µl of 2 mM DTSSP (Pierce) freshly made in PBS was added. Cross-linking reactions were allowed to proceed for 15 min on ice and terminated by the addition of 1 ml of 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris (pH 7.4), 0.5% sodium deoxycholate, 50 mM NaF, and 1 mM PMSF). The lysates were used in immunoprecipitation and immunoblotting as previously described (39). Samples were boiled in SDS sample buffer without or with 1% {beta}-ME (Sigma-Aldrich, St. Louis, MO) for 4 min before loading to the gel for SDS-PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMA treatment partially restored the surface CD4 staining in T cells expressing Nef-GFP

Because both Nef and PMA can down-regulate CD4, apparently by two independent mechanisms, we started this study by asking the question whether Nef and PMA were synergistic in CD4 down-regulation. We transfected Jurkat T cells with Nef-GFP. The cells were previously stably transfected with wt CD4, 2C CD4 (a non-Lck binding CD4), or tail-less CD4 as previously described (39). After PMA treatment, cells were surface-stained with the PE-conjugated anti-CD4 for two-color cytometric analysis (Fig. 1a). FL1 shows the GFP fluorescence intensity of Nef-GFP expression; FL2 shows the CD4 surface staining. Without PMA treatment (–PMA), Nef-GFP down-regulated wt CD4 and 2C CD4 in a dose-dependent manner. In cells expressing high levels of Nef-GFP (FL1, >500), CD4 was almost completely down-regulated. Unexpectedly, with PMA treatment (+PMA), the Nef down-regulated wt CD4 and 2C CD4 were partially restored to the cell surface in Nef-GFP-positive cells (FL1 >10; right to the vertical line). In contrast, CD4 was down-regulated by PMA in Nef-GFP-negative cells (FL1, <10).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 1. PMA treatment partially restored the surface CD4 expression in Jurkat T cells expressing Nef-GFP. Jurkat T cells stably expressing wt CD4, 2C CD4, or tail-less CD4 were transfected with Nef-GFP. Eighteen hours after transfection, cells were treated with PMA and then stained with anti-CD4 PE for FACS. a, Two-color cytometric analysis of the surface CD4 staining. The x-axis (FL1-H) shows the fluorescence intensity of Nef-GFP; the y-axis (FL2-H) shows the CD4-PE staining. See Materials and Methods for the line settings in FACS. b, Effects of PMA on relative surface expression of CD4 in Nef-GFP positive cells (FL1, >10) or Nef-GFP negative cells (FL1, <10). The percentage is calculated from the FACS results shown in a as described in Materials and Methods. c, Time course and dose dependence of the surface CD4 staining treated with PMA. Nef-GFP-transfected cells were treated with different doses of PMA for 20 min at 37°C or treated with 100 ng/ml PMA for different time periods. The percentage of CD4 staining was calculated from the FACS results.

 
For a better comprehension, Fig. 1b quantitatively compared the effects of PMA on the surface CD4 expression in Nef-positive or Nef-negative cells. The left panel shows the percentage of surface CD4 expression in Nef-negative cells. PMA treatment reduced the surface CD4 of wt CD4 and 2C CD4 by 40 and 75%, respectively (converting the results in Fig. 1a from a log scale to a linear scale). The right panel shows the percentage of surface CD4 in Nef-positive cells. Without PMA treatment, Nef-GFP down-regulated wt CD4 and 2C CD4 to 5 and 3% of the steady state level (medium CD4 staining without PMA treatment). With PMA treatment, the surface wt CD4 and 2C CD4 were restored from 5 to 35% and from 3 to 20%, respectively. The tail-less CD4 was not down-regulated by PMA treatment or by Nef expression (Fig. 1, a and b), in agreement with the observations by others that CD4 tail was necessary for the PMA-induced (3) and the Nef-dependent CD4 down-regulation (21, 22). The surface expression of tail-less CD4 in Nef-expressing cells was also not affected by PMA treatment, suggesting that the CD4 tail may be the part in CD4 that participated in the restoration.

The dose of PMA and the time required for the restoration were determined (Fig. 1c). PMA at 10–20 ng/ml was sufficient, and the process started right after the addition of PMA and completed within 10 min, with kinetics similar to those of PMA activation of PKC.

Partial restoration of surface CD4 by PMA may require the phosphorylation of CD4 at Ser408 and Ser415

It is known that the PMA-induced phosphorylation of Ser408 and Ser415 in the CD4 tail is necessary for the down-regulation of CD4 (3, 4). To determine whether the phosphorylation of Ser408 and Ser415 was also involved in the partial restoration of the surface CD4 in Nef-expressing T cells, we generated a serine CD4 mutant (Ser CD4), in which Ser408 and Ser415 were replaced by alanine. T cells stably expressing Ser CD4 were transfected with Nef-GFP. Serine mutation did not affect the Nef-induced down-regulation of CD4 (Fig. 2a), in agreement with the previous report (12). The extent of the down-regulation of Ser CD4 by Nef-GFP was similar to that of wt CD4 (Fig. 2a, left panel). When treated with PMA (Fig. 2a, right panel), however, unlike wt CD4, the surface staining of Ser CD4 was not restored. Moreover, the restoration of the surface staining of wt CD4 by PMA was abolished when Jurkat T cells were preincubated with a PKC inhibitor, BIM1, at 50–100 nM (Fig. 2b). The results thus suggested that the PMA-induced phosphorylation of Ser408 and Ser415 was required for the restoration of the surface CD4 in Nef-GFP-expressing T cells.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. The phosphorylation of CD4 at Ser408 and Ser415 is required for the restoration of the surface CD4 in Nef-GFP-expressing T cells. a, Restoration of wt CD4 and Ser CD4 in cells treated with PMA. Cells expressing wt CD4 or Ser CD4 were transfected with Nef-GFP for 18 h. After PMA treatment, cells were stained with CD4-PE for FACS. b, Inhibition of the CD4 restoration by PKC inhibitor BIM1. Nef-GFP-transfected cells were incubated with BIM1 at different concentrations for 5 min at 37°C. Cells were then treated with PMA and stained with CD4-PE. The percentage of CD4 staining was calculated from the FACS results as described in Fig. 1b.

 
PMA triggered a reversal of the Nef down-regulation of CD4

A simple explanation of the partial restoration of CD4 by PMA is the reversal of Nef down-regulation of CD4, possibly at the assembly step of the CD4-Nef-AP complex. Therefore, we examined the effects of PMA on CD4 down-regulation in cells expressing Nef-GFP mutants defective for linking CD4 to the AP complex. In NefWL-GFP mutant, residues W57 and L58 essential for Nef interaction with CD4 were substituted by alanines. In NefLL-GFP mutant, the dileucine motif (L164-L165) essential for Nef interaction with an AP complex was similarly substituted. FACS analysis indicated that, in contrast to wt Nef-GFP (Fig. 3, upper left panel), NefWL-GFP and NefLL-GFP did not down-regulate CD4 (Fig. 3, middle left and lower left panels), excluding a few highly NefWL-GFP-positive cells (FL1, >500) where there was a low level of CD4 down-regulation. Unlike the case with wt Nef-GFP (upper right panel), PMA treatment did not increase surface CD4 that was not down-regulated in cells expressing NefWL-GFP and NefLL-GFP (Fig. 3, middle right and lower right panels), excluding a few highly NefWL-GFP-positive cells where the surface CD4 staining was partially restored. The results indicated that only when CD4 was down-regulated by Nef-GFP was PMA able to partially restore the surface staining of CD4, suggesting that PMA triggered a reversal of the Nef down-regulation of CD4, probably by affecting the CD4-Nef interaction, because PMA-induced phosphorylation of the CD4 tail may alter its conformation (3, 4).



View larger version (59K):
[in this window]
[in a new window]
  		FIGURE 3. Effects of the expression of NefLL and NefWL mutants on PMA-induced CD4 internalization. Cells expressing wt CD4 were transfected with Nef-GFP, NefLL-GFP, and NefWL-GFP for 18 h; treated with PMA; then stained with CD4-PE for FACS.

 
Cell fractionation and chemical cross-linking showed that PMA treatment disrupted the interaction between CD4 and Nef

To determine whether PMA-induced phosphorylation of the CD4 tail disrupts the CD4-Nef interaction, we set out to show that there was an association between CD4 and Nef in T cells. Before our study, attempts to coimmunoprecipitate wt Nef and CD4 in samples from mammalian cells had failed (24). Therefore, we tried to coimmunoprecipitate CD4 and NefLL, because NefLL interacts with, but does not down-regulate, CD4. To our disappointment, like wt Nef, NefLL too was not coimmunoprecipitated with CD4 (complete data not shown; cf., Fig. 4b, lane 2). We then resorted to cell fractionation and chemical cross-linking. Cells were cotransfected with constructs expressing GFP and each of the three non-GFP-tagged Nef (wt Nef, NefLL, and NefWL). The use of the nontagged Nef was necessary to avoid a possible nonspecific cross-linking between CD4 and the GFP-tag. Approximately 15–20% of the cells expressed Nef according to the expression of the cotransfected GFP (data not shown). The cells were then fractionated into membrane and cytosol fractions. The immunoblotting analysis showed that CD4 and all three Nef were predominantly membrane-associated (Fig. 4a, –PMA). Anti-CD4 blotting (Fig. 4a, upper panel) showed that the membrane-associated CD4 was ~20% less in cells transfected with wt Nef (lane 6) than in cells mock-transfected (lane 5), NefLL-transfected (lane 7), or NefWL-transfected (lane 8; analyzed by densitometry). Considering that only 15–20% cells expressed Nef, the results indicated that CD4 was down-regulated by wt Nef, but not by NefLL or NefWL, in agreement with the FACS results with expression of GFP-tagged Nef (Fig. 3). PMA treatment increased the CD4 level in the cytosol fractions (Fig. 4a, lanes 9–12 vs lanes 1–4) and decreased the CD4 level in the membrane fractions (Fig. 4a, lanes 5–8 vs lanes 13–16). Also, PMA treatment attenuated the difference in membrane-associated CD4 amounts between cells transfected with wt Nef and Nef mutants (lanes 13–16). The results suggested a reversal of Nef down-regulation of CD4 by PMA in agreement with the results by FACS (Fig. 1). PMA treatment essentially did not affect the membrane distribution of Nef, including wt Nef, NefLL, and NefWL (Fig. 4a, bottom panel). At least 3–4 times more NefLL (lanes 3, 7, 11, and 15) than wt Nef (lanes 2, 6, 10, and 14) and NefWL (lanes 4, 8, 12, and 16) was detected, even though cells were transfected with equal amounts of different plasmid DNAs.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 4. Effects of PMA on membrane distribution and chemical cross-linking of CD4 and Nef mutants. Cells were transfected with nontagged wt Nef (WT), NefLL(LL), or NefWL(WL) or were mock-transfected (–), treated with PMA, and then fractionated into membrane and cytosolic fractions. a, Anti-CD4 and anti-Nef immunoblotting of cytosol and membrane fractions. The amounts of cytosol and membrane fractions represented 1/30th and 1/6th of each treated cells, respectively. Cells were expressing wt CD4. After protein transfer, the polyvinylidene difluoride membrane was cut into two pieces. The top part was immunoblotted with polyclonal anti-CD4 (top panel); the bottom was immunoblotted with anti-Nef rabbit antiserum (bottom panel). b, The membrane fractions were cross-linked by DTSSP, except in lane 2, and then lysed in Nonidet P-40 lysis buffer. After immunoprecipitation with anti-CD4, the samples were immunoblotted with anti-CD4 or anti-Nef as indicated. Cells were mock-transfected (lane 1), transfected with NefLL (lanes 2–7, 8, 10, 12, and 14) or with wt Nef (lanes 9, 11, 13, and 15). T cells expressed wt CD4 (lanes 1–3 and 5–11), tail-less CD4 (lane 4), or Ser CD4 (lanes 12–15). Twice the amount of membrane from PMA-treated wt CD4 cells (lanes 11 and 12) as from NefLL was immunoprecipitated with anti-CD4. Samples were run in nonreducing conditions, except in lane 7. c, Anti-Nef immunoblotting of the nontagged Nef protein (lanes 2–4) and Nef-GFP protein (lanes 5–7). Cells (107) were transfected with 20 µg of plasmid DNA and lysed in 100 µl of Nonidet P-40 lysis buffer. Lysates (25 µl) was resolved by SDS-PAGE, then immunoblotted with anti-Nef.

 
For chemical cross-linking experiments, we used the membrane fraction only because the CD4-Nef interaction occurred on the T cell membrane (25). In addition to the membrane fraction from the wt Nef-transfected cells, we used the membrane fraction from the NefLL-transfected cells, because among wt Nef and two Nef mutants, it was expressed at the highest level, and it was defective in Nef-AP interaction, which would make the interpretation of results less complicated than for the wt Nef. The wt Nef and NefLL were transfected into cells expressing wt CD4, tail-less CD4, or Ser CD4. After fractionation, the membrane fractions were treated with DTSSP, a thiol-cleavable cross-linker. The samples were then immunoprecipitated and analyzed by immunoblotting (Fig. 4b). Anti-CD4 recognized a protein band of ~80–85 kDa on nonreducing SDS gel from the NefLL-transfected samples treated with DTSSP (lanes 3 and 5), but not from the mock-transfected samples (lane 1) or sample not treated with DTSSP (lane 2). Anti-Nef also recognized the 80- to 85-kDa band on nonreducing SDS gel, but recognized a 27-kDa band on reducing SDS gel, which was the cleaved product of the 80- to 85-kDa band by {beta}-ME and corresponded to the size of Nef (lane 7). The results thus indicated that the 80- to 85-kDa band was the chemically cross-linked CD4-Nef product. Tail-less CD4 was not cross-linked (lane 4), indicating that the CD4 tail was required for the interaction.

The effects of PMA on the chemical cross-linking are shown in the right panel of Fig. 4b. From samples transfected with wt CD4, the 80- to 85-kDa cross-linking products (lanes 8 and 9) disappeared upon PMA treatment (lanes 10 and 11). In contrast, from samples transfected with the nonphosphorylatable Ser CD4, the similar 80- to 85-kDa band was detected regardless of PMA treatment (lanes 12–15). The results thus suggested that the PMA-induced serine phosphorylation of CD4 might disrupt the interaction between CD4 and Nef. Note that the 80- to 85-kDa band for NefLL (lanes 8, 12, and 14) was more intense than that for wt Nef (lanes 9, 13, and 15), consistent with the above observation that more NefLL than wt Nef was present on T cell membrane (Fig. 4a).

By anti-Nef immunoblotting, the amount of nontagged NefLL in the membrane fraction was ~3- to 4-fold more than that of wt Nef or NefWL (Fig. 4a), whereas by FACS on GFP fluorescence, similar amounts of wt Nef-GFP, NefLL-GFP, and NefWL-GFP were detected (Fig. 3). To verify that tagging GFP to Nef made the disappearance of the overexpression of NefLL relative to the other two Nef, we side-by-side compared the expression levels of nontagged and GFP-tagged Nef by anti-Nef immunoblotting (Fig. 4c). Again, no difference was found among Nef-GFP, whereas NefLL was ~3- to 4-fold more than the wt Nef and NefWL. Therefore, the phenomenon proved true. A plausible explanation might be that the dileucine motif in Nef might be involved in Nef endocytosis and/or degradation and that tagging GFP to Nef might slow down Nef degradation to the extent that the difference in the amounts of the three GFP-tagged Nef becomes greatly diminished. This possibility is currently under further investigation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we found that HIV Nef-down-regulation of CD4 from the Jurkat T cell surface was reversible by PMA treatment (Fig. 1). The reversal appeared to require the phosphorylation of CD4, because both mutating Ser408 and Ser415, the PMA phosphorylation sites in CD4 (3, 4), and the use of a PKC inhibitor, BIM1, abolished the reversal (Fig. 2). CD4 and Nef could be cross-linked by DTSSP in membranes isolated from T cells not treated with PMA, but not in membranes from PMA-treated cells (Fig. 4). Taken together, the data suggested that CD4 and Nef were associated with each other in T cells, and the Ser phosphorylation of CD4 disrupted the interaction.

It has been shown that the CD4-associated Nef binds to the {beta}-coatomer protein on endosomes, targeting CD4 for lysosomal degradation (40). As a result, Nef prevents down-regulated CD4 molecules from recycling back to the cell surface. In the absence of Nef-{beta}-coatomer protein interaction, ~45% of internalized CD4 molecules recycled back to the cell surface within 10 min (40). This explains why the magnitude of CD4 down-regulation by Nef is more complete than that by PMA. In this study, for instance, CD4 down-regulation by Nef was nearly complete (95%), whereas that by PMA was partial (40%; Fig. 1, a and b).

A postulated mechanism of the reversal is as follows. The Nef down-regulation of CD4 is mediated through the CD4-Nef interaction. PMA induces phosphorylation of the CD4 tail, which disrupts the CD4-Nef interaction. The phosphorylated CD4 tail now bypasses Nef, binding directly to an AP complex, most likely to AP-2 (our preliminary data not shown). In other words, PMA treatment switches CD4 internalization from a Nef-dependent to a Nef-independent pathway, which allows some of the down-regulated CD4 molecules to recycle back to the cell surface, resulting in a net gain in cell surface CD4 in Nef-expressing T cells.

Our hypothesis is consistent with the known facts about CD4 and Nef. The CD4 tail comprises a single {alpha}-helix from Arg402 to Lys417 (41). Leu413, Leu414, Ser408, and Ser415 are all part of this {alpha}-helix. The dileucine motif (Leu413 and Leu414) involved in the CD4-Nef interaction and CD4 internalization is close to Ser408 and Ser415. Therefore, the phosphorylation of Ser408 and Ser415 may directly disrupt the CD4-Nef interaction or may indirectly do so by altering the conformation of the CD4 tail. In this respect, it is known that the phosphorylation of Ser408 and Ser415 of CD4 results in the dissociation of Lck from the CD4 tail as a result of a conformational change in the CD4 tail (5). It is conceivable that the same conformational change that breaks the interaction between CD4 and Lck may also break the interaction between CD4 and Nef. Consistent with this is the observation that Lck and Nef appear to complete for binding to CD4 (42).


   Acknowledgments
 
We thank J. Skowronski for the Nef-GFP constructs. We also thank John Hirst for FACS analysis.


   Footnotes
 
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.

1 This work was supported in part by grants from National Institutes of Health, the Association for International Cancer Research (United Kingdom), and a pilot AIDS funding from New York University. Back

2 Address correspondence and reprint requests to Dr. Yong-Jiu Jin, Skirball Institute of Biomedical Research, 540 1st Avenue, Lab 5-1, New York University School of Medicine, New York, NY 10016. E-mail address: jiny{at}saturn.med.nyu.edu Back

3 Abbreviations used in this paper: PKC, protein kinase C; AP, adaptor protein complex; BIM1, bisindolylmaleimide 1; DTSSP, 3,3-dithiobis[sulfosuccinimidyl-propionate]; wt, wild type. Back

Received for publication June 29, 2004. Accepted for publication August 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pelchen-Matthews, A., J. E. Armes, M. Marsh. 1989. Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells. EMBO J. 8:3641.[Medline]
  2. Shin, J., C. Doyle, Z. Yang, D. Kappes, J. L. Strominger. 1990. Structural features of the cytoplasmic region of CD4 required for internalization. EMBO J. 9:425.[Medline]
  3. Shin, J., R. L. Dunbrack, Jr, S. Lee, J. L. Strominger. 1991. Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4. J. Biol. Chem. 266:10658.[Abstract/Free Full Text]
  4. Pitcher, C., S. Honing, A. Fingerhut, K. Bowers, M. Marsh. 1999. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. Cell 10:677.[Abstract/Free Full Text]
  5. Pelchen-Matthews, A., I. J. Parsons, M. Marsh. 1993. Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting. J. Exp. Med. 178:1209.[Abstract/Free Full Text]
  6. Marsh, M., A. Pelchen-Matthews. 1996. Endocytic and exocytic regulation of CD4 expression and function. Curr. Top. Microbiol. Immunol. 205:107.[Medline]
  7. Cullen, B. R.. 1994. The role of Nef in the replication cycle of the human and simian immunodeficiency viruses. Virology 205:1.[Medline]
  8. Kestler, H. W., III, D. J. Ringler, K. Mori, D. L. Panicali, P. K. Sehgal, M. D. Daniel, R. C. Desrosiers. 1991. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651.[Medline]
  9. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J. Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, et al 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270:988.[Abstract/Free Full Text]
  10. Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, R. C. Desrosiers. 1995. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332:228.[Free Full Text]
  11. Learmont, J. C., A. F. Geczy, J. Mills, L. J. Ashton, C. H. Raynes-Greenow, R. J. Garsia, W. B. Dyer, L. McIntyre, R. B. Oelrichs, D. I. Rhodes, et al 1999. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1: a report from the Sydney Blood Bank Cohort. N. Engl. J. Med. 340:1715.[Abstract/Free Full Text]
  12. Garcia, J. V., A. D. Miller. 1991. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350:508.[Medline]
  13. Benson, R. E., A. Sanfridson, J. S. Ottinger, C. Doyle, B. R. Cullen. 1993. Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection. J. Exp. Med. 177:1561.[Abstract/Free Full Text]
  14. Mariani, R., J. Skowronski. 1993. CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals. Proc. Natl. Acad. Sci. USA 90:5549.[Abstract/Free Full Text]
  15. Lama, J., A. Mangasarian, D. Trono. 1999. Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation in a Nef- and Vpu-inhibitable manner. Curr. Biol. 9:622.[Medline]
  16. Ross, T. M., A. E. Oran, B. R. Cullen. 1999. Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein. Curr. Biol. 9:613.[Medline]
  17. Glushakova, S., J. Munch, S. Carl, T. C. Greenough, J. L. Sullivan, L. Margolis, F. Kirchhoff. 2001. CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4+ T-cell depletion in human lymphoid tissue ex vivo. J. Virol. 75:10113.[Abstract/Free Full Text]
  18. Cortes, M. J., F. Wong-Staal, J. Lama. 2002. Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells. J. Biol. Chem. 277:1770.[Abstract/Free Full Text]
  19. Lundquist, C. A., M. Tobiume, J. Zhou, D. Unutmaz, C. Aiken. 2002. Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J. Virol. 76:4625.[Abstract/Free Full Text]
  20. Stoddart, C. A., R. Geleziunas, S. Ferrell, V. Linquist-Stepps, M. E. Moreno, C. Bare, W. Xu, W. Yonemoto, P. A. Bresnahan, J. M. McCune, et al 2003. Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis. J. Virol. 77:2124.[Abstract/Free Full Text]
  21. Aiken, C., J. Konner, N. R. Landau, M. E. Lenburg, D. Trono. 1994. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76:853.[Medline]
  22. Anderson, S. J., M. Lenburg, N. R. Landau, J. V. Garcia. 1994. The cytoplasmic domain of CD4 is sufficient for its down-regulation from the cell surface by human immunodeficiency virus type 1 Nef. J. Virol. 68:3092.[Abstract/Free Full Text]
  23. Mangasarian, A., M. Foti, C. Aiken, D. Chin, J. L. Carpentier, D. Trono. 1997. The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane. Immunity 6:67.[Medline]
  24. Arora, V. K., B. L. Fredericksen, J. V. Garcia. 2002. Nef: agent of cell subversion. Microbes Infect. 4:189.[Medline]
  25. Harris, M. P., J. C. Neil. 1994. Myristoylation-dependent binding of HIV-1 Nef to CD4. J. Mol. Biol. 241:136.[Medline]
  26. Grzesiek, S., S. J. Stahl, P. T. Wingfield, A. Bax. 1996. The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef: mapping of the Nef binding surface by NMR. Biochemistry 35:10256.[Medline]
  27. Mangasarian, A., V. Piguet, J. K. Wang, Y. L. Chen, D. Trono. 1999. Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal {alpha} helix and proline repeat of Nef selectively regulate MHC-I trafficking. J. Virol. 73:1964.[Abstract/Free Full Text]
  28. Rossi, F., A. Gallina, G. Milanesi. 1996. Nef-CD4 physical interaction sensed with the yeast two-hybrid system. Virology 217:397.[Medline]
  29. Preusser, A., L. Briese, A. S. Baur, D. Willbold. 2001. Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain. J. Virol. 75:3960.[Abstract/Free Full Text]
  30. Greenberg, M., L. DeTulleo, I. Rapoport, J. Skowronski, T. Kirchhausen. 1998. A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. Curr. Biol. 8:1239.[Medline]
  31. Bresnahan, P. A., W. Yonemoto, W. C. Greene. 1999. Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4. J. Immunol. 163:2977.[Abstract/Free Full Text]
  32. Craig, H. M., M. W. Pandori, J. C. Guatelli. 1998. Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. Proc. Natl. Acad. Sci. USA 95:11229.[Abstract/Free Full Text]
  33. Bonifacino, J. S., L. M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395.[Medline]
  34. Robinson, M. S.. 2004. Adaptable adaptors for coated vesicles. Trends Cell. Biol. 14:167.[Medline]
  35. Greenberg, M. E., S. Bronson, M. Lock, M. Neumann, G. N. Pavlakis, J. Skowronski. 1997. Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation. EMBO J. 16:6964.[Medline]
  36. Craig, H. M., T. R. Reddy, N. L. Riggs, P. P. Dao, J. C. Guatelli. 2000. Interactions of HIV-1 nef with the µ subunits of adaptor protein complexes 1, 2, and 3: role of the dileucine-based sorting motif. Virology 271:9.[Medline]
  37. Janvier, K., Y. Kato, M. Boehm, J. R. Rose, J. A. Martina, B. Y. Kim, S. Venkatesan, J. S. Bonifacino. 2003. Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 {gamma}-{sigma}1 and AP-3 {delta}-{sigma}3 hemicomplexes. J. Cell Biol. 163:1281.[Abstract/Free Full Text]
  38. Bresnahan, P. A., W. Yonemoto, S. Ferrell, D. Williams-Herman, R. Geleziunas, W. C. Greene. 1998. A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor. Curr. Biol. 8:1235.[Medline]
  39. Fragoso, R., D. Ren, X. Zhang, M. W. Su, S. J. Burakoff, Y. J. Jin. 2003. Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling. J. Immunol. 170:913.[Abstract/Free Full Text]
  40. Piguet, V., F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, D. Trono. 1999. Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of {beta}-COP in endosomes. Cell 97:63.[Medline]
  41. Wray, V., D. Mertins, M. Kiess, P. Henklein, W. Trowitzsch-Kienast, U. Schubert. 1998. Solution structure of the cytoplasmic domain of the human CD4 glycoprotein by CD and 1H NMR spectroscopy: implications for biological functions. Biochemistry 37:8527.[Medline]
  42. Salghetti, S., R. Mariani, J. Skowronski. 1995. Human immunodeficiency virus type 1 Nef and p56lck protein-tyrosine kinase interact with a common element in CD4 cytoplasmic tail. Proc. Natl. Acad. Sci. USA 92:349.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y.-J. Jin, C. Y. Cai, X. Zhang, and S. J. Burakoff
Lysine 144, a Ubiquitin Attachment Site in HIV-1 Nef, Is Required for Nef-Mediated CD4 Down-Regulation
J. Immunol., June 15, 2008; 180(12): 7878 - 7886.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
J. F. Roeth and K. L. Collins
Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways
Microbiol. Mol. Biol. Rev., June 1, 2006; 70(2): 548 - 563.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y.-J. Jin, C. Y. Cai, X. Zhang, H.-T. Zhang, J. A. Hirst, and S. J. Burakoff
HIV Nef-Mediated CD4 Down-Regulation Is Adaptor Protein Complex 2 Dependent
J. Immunol., September 1, 2005; 175(5): 3157 - 3164.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jin, Y.-J.
Right arrow Articles by Burakoff, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jin, Y.-J.
Right arrow Articles by Burakoff, S. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS