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Protein Kinase C Is Involved in the Regulation of Both Signaling and Adhesion Mediated by DNAX Accessory Molecule-1 Receptor

Department of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304

	Abstract

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DNAX accessory molecule-1 (DNAM-1) is a signal-transducing adhesion molecule involved in the cytolytic function mediated by CTL and NK cells. In the present study, we have investigated various perimeters of DNAM-1-mediated signaling and adhesion. Although adhesion of DNAM-1 to its ligand does not require divalent cations, protein synthesis, or RNA transcription, activation of protein kinase C (PKC) is required for DNAM-1 functioning. Furthermore, mutation of the putative PKC-binding site in the cytoplasmic domain of DNAM-1 (Ser³²⁹ to Phe³²⁹) prevents both ligand binding and PMA-induced phosphorylation of the DNAM-1 receptor. These results indicate that PKC phosphorylates Ser³²⁹ of DNAM-1 and plays a critical role for both DNAM-1 adhesion and signaling.

	Introduction

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Intercellular adhesion molecules (ICAMs) play an important role in lymphocyte-mediated immune responses (1, 2, 3, 4, 5, 6, 7). Cell adhesion molecules provide not only for intercellular binding, but also participate in signal transduction (2, 8). A variety of cell adhesion molecules, including the members of the cadherin, integrin, Ig, and selectin families, are involved in this process (8, 9).

One of the most interesting characteristics of adhesion molecules is their dynamic role in adhesion and detachment. Lymphocytes rapidly interconvert between a nonadherent state in circulation and an adherent state in tissues. Although the regulation of adhesion is not well understood, lymphocyte activation and structural features of the cytoplasmic domain of adhesion molecules play important roles in triggering and/or facilitating intercellular binding. For example, interaction of LFA-1 with its ligands, the ICAMs, is augmented by cell activation initiated by signals from TCR (5, 10). Upon stimulation, LFA-1 avidity for ICAM-1 is transiently increased over a period of minutes. Structural studies have determined that the three contiguous threonines within the cytoplasmic domain of the ß subunit of LFA-1 are critical for binding to ICAM-1 (11, 12).

We have recently described an adhesion molecule, DNAX accessory molecule-1 (DNAM-1),³ which is expressed on the majority of T cells and NK cells (13). DNAM-1 is a type 1 transmembrane glycoprotein and a member of the Ig supergene family. Cross-linking DNAM-1 with anti-DNAM-1 mAb induces cytolysis mediated by CTL and NK cells and also results in tyrosine phosphorylation of the DNAM-1 molecule, indicating that DNAM-1 transduces an activation signal. COS-7 cells transfected with DNAM-1 bind to a colon carcinoma cell line, Colo-205, and this binding is specifically blocked by anti-DNAM-1 mAb, suggesting that Colo-205 expresses a cell surface ligand (DNAM-1L). We have now investigated the structural features of the DNAM-1 receptor that are involved in adhesion and signal transduction. We report here that PKC phosphorylates Ser³²⁹ of DNAM-1 and plays a critical role for both DNAM-1 adhesion and signaling.

	Materials and Methods

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Cell lines and reagents

P815 and BW5147 are mouse mastocytoma and thymoma cell lines, respectively. Colo-205 is a human colon carcinoma cell line. PMA, paraformaldehyde, cyclohexamide, and actinomycin D were purchased from Sigma (St. Louis, MO). PKC inhibitor GF109203X was purchased from Calbiochem (San Diego, CA). PMA, GF109203X, and actinomycin D were dissolved in DMSO (which did not exceed 0.1% in the assay medium). A 1% paraformaldehyde solution was prepared in PBS (pH 7.4).

NK clones

NK clones were established from peripheral blood of healthy donors using the culture conditions described previously (14) with 100 IU/ml rIL-2 as a growth factor.

Cytotoxicity assay

Tumor cell lines (1.5 x 10⁶ cells) were labeled with 100 µCi ⁵¹Cr for 2 h, washed, and used as targets in a 4-h radioisotope release cytotoxicity assay, as described (15). Data are expressed as the means of triplicate cultures, and spontaneous radioisotope release was typically <10% of total ⁵¹Cr release. Percent cytolysis was calculated as:

mAbs and flow cytometry

Control IgG was generously provided by Becton Dickinson Immunocytometry Systems (San Jose, CA). The anti-monomorphic HLA class I mAb (DX17 mAb) (16) and anti-DNAM-1 mAb (DX11 mAb) (13) were generated as described. Methods of immunofluorescent staining and flow cytometry have been described previously (17).

Establishment of transfectants of BW5147, which stably express DNAM-1

Wild-type and several deletion mutant DNAM-1 cDNA were produced by PCR using the DNAM-1 cDNA plasmid LL378 (13) as a template. To generate a site-specific DNAM-1 mutant at residue 329, an antisense PCR primer, which contained a codon for Phe³²⁹ (TTT) instead of Ser³²⁹ (TCT), was designed. The PCR products were subcloned into a retroviral vector pMX-neo (kindly provided by Dr. Toshio Kitamura) with cloning sites of BamHI (5') and Not1 (3'). BOSC23 packaging cells (18) were transfected with the DNAM-1 cDNA in the retroviral vector using lipofectamine (Life Technologies, Gaithersburg, MD) as described previously (19, 20). BW5147 cells were infected with the DNAM-1 retrovirus stock. Two days after infection, the cells were analyzed and DNAM-1-expressing cells were cloned by flow cytometry. All mutant cDNAs were verified by sequencing.

Cell adhesion assay

The BW5147 transfectants were incubated in PBS containing 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (Boehringer Mannheim Biochemica, Mannheim, Germany) at the concentration of 0.4 µg/ml for 1 h at room temperature and then washed with PBS three times, so that BW5147 transfectants fluoresced green. Colo-205 cells were stained with phycoerythrin (PE)-conjugated anti-human HLA class I (DX17 mAb) and then washed with PBS three times, so that Colo-205 cells fluoresce red. A total of 5 x 10⁵ BW5147 transfectants and 1 x 10⁵ Colo-205 cells were suspended in 200 µl of RPMI 1640 medium with 10% FCS containing or lacking EGTA and/or EDTA (each concentration at 0.5 mM), or in 200 µl HEPES buffer (0.13 M NaCl, 10 mM HEPES, 10 mM glucose, and 0.5% BSA) containing or lacking both calcium and magnesium (each concentration at 1.0 mM), and mixed in a 12- x 75-mm polystyrene tube (Becton Dickinson Labware, Franklin Lakes, NJ). The cell mixture was centrifuged at 500 rpm (40 x g) for 3 min and incubated at 37°C, room temperature, or 4°C. The cells were then gently resuspended and analyzed by flow cytometry.

In some experiments, green fluorochrome-labeled BW5147 transfectants were pretreated with DMSO alone (0.1%), cyclohexamide (10 µg/ml), actinomycin D (10 µg/ml), PMA (50 ng/ml), or GF109203X at the indicated concentrations for 2 h at 37°C. In experiments to determine the metabolic requirements for DNAM-1 binding, BW5147 transfectants or Colo-205 cells were fixed with 1% paraformaldehyde PBS for 1 h at 4°C and washed extensively with PBS (21). The transfectants were cocultured with Colo-205 (stained with PE-conjugated anti-HLA class I mAb) and analyzed by flow cytometry, as described above. The percentage of Colo-205 cells binding to the BW5147 transfectants was calculated as follows:

Biochemistry

Cells were labeled with [³²P]orthophosphate (Amersham Corp., Arlington Heights, IL) and stimulated or not with PMA (50 ng/ml, 2 h at 37°C). Cells were then lysed in Tris-buffered saline (50 mM Tris, 15 mM NaCl, pH 8.0) containing 1% Nonidet P-40 and protease inhibitors (1 mM PMSF and 20 Kallikrein inhibitor U/ml aprotinin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM Na₄P₂O₇, 0.1 mM ß-glycerophosphate, and 1 mM Na₃VO₄). DNAM-1 Ag was immunoprecipitated using the method described previously (22). Samples were analyzed by SDS-PAGE and radioisotopes detected using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

	Results

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DNAM-1 adhesion is temperature dependent, but does not require divalent cations, protein synthesis, or RNA transcription

In order to study the regulation of DNAM-1 adhesion, we established a transfectant of the mouse T cell line BW5147 which stably expresses human DNAM-1 (Fig. 1A). Using this transfectant and the colon carcinoma Colo-205, which expresses a DNAM-1L (13), we developed a cell-cell adhesion assay to determine the requirements for DNAM-1 binding. As shown in Figure 1B, 54% of Colo-205 cells bound to the DNAM-1 BW5147 transfectant after incubation for 60 min at 37°C, whereas <10% of Colo-205 cells bound to untransfected BW5147 cells. The binding of Colo-205 to DNAM-1⁺ BW5147 cells was specifically inhibited when the transfectants were preincubated with DX11 mAb (Fig. 1B), demonstrating specificity of the interaction. Equivalent binding was observed in medium containing or lacking both calcium and magnesium (each concentration at 1.0 mM). Additionally, DNAM-1 binding was not affected when EDTA or EGTA were present in the medium (not shown). These results indicate that DNAM-1L ligand binding does not require divalent cations. When Colo-205 and the DNAM-1 transfectant were cocultured at room temperature or 4°C, the binding was significantly less than at 37°C (Fig. 1C). These results suggest that efficient DNAM-1 adhesion to DNAM-1L may require a metabolic change of either DNAM-1, DNAM-1L, or both molecules. To determine which molecule was affected, Colo-205 and/or the DNAM-1 transfectant were fixed with paraformaldehyde and then cocultured at 37°C. Colo-205 binding to DNAM-1 transfectants was significantly inhibited when the DNAM-1 transfectants, but not Colo-205 cells, were fixed with paraformaldehyde (Fig. 1C), suggesting that the DNAM-1 receptor is either sensitive to fixation or may undergo a conformational alteration to permit ligand binding. Binding was not affected by pretreatment of DNAM-1 transfectants with either cyclohexamide (inhibitor of protein synthesis) or actinomycin D (inhibitor of transcription) (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Effect of divalent cations and metabolic changes on DNAM-1 adhesion. A, Establishment of BW5147 transfectant with wild-type DNAM-1. The transfectants were stained with either PE-conjugated control IgG (open histograms) or PE-conjugated anti-DNAM-1 mAb DX11 (shaded histograms), and analyzed by flow cytometry. The transfectants stably express the wild-type DNAM-1 Ag. B, DNAM-1 adhesion assay. Human colon carcinoma cell Colo-205 was stained with PE-conjugated anti-human HLA class I mAb (DX17) so that these cells would fluoresce red, detected on FL-2. Mouse T cell BW5147 or the DNAM-1+ BW5147 transfectants were stained with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester so that these cells would fluoresce green, detected on FL-1. Colo-205 and transfectants were cocultured in RPMI 1640 medium with 10% FCS at 37°C for 1 h and analyzed by flow cytometry at the dimension of FSC and SSC (upper panel) and FL-1 and FL-2 (lower panel). Aggregates of cells containing both Colo-205 and BW cells were scored as double positive (upper right quadrant). Note that Colo-205 cells show significant binding to the DNAM-1+ BW5147 transfectant (middle), but do not bind to untransfected BW5147 cells (left). Binding of DNAM-1+ transfectants to Colo-205 was inhibited when the transfectants were precoated with DX11 mAb (right). C, The effect of temperature and fixation on DNAM-1 adhesion. Upper panel, The adhesion assay was performed at 37°C, room temperature, or 4°C in RPMI 1640 medium with 10% FCS for 1 h. Lower panel, DNAM-1+ BW 5147 cells and/or Colo-205 cells were fixed with 1% paraformaldehyde and assayed for adhesion. Fixation of DNAM-1+ BW5147 cells, but not Colo-205, inhibited the binding of Colo-205 to the transfectant.

We have previously reported that anti-DNAM-1 mAb DX11 activated the cytolytic activity of CTL when the CTL were cocultured with the murine FcR-bearing target P815 mastocytoma (13). DX11 mAb, as well as anti-CD16 (FcRIII), induced redirected cytolysis by NK clones against P815. Since NK cell-mediated cytotoxicity, but not Ab-dependent cellular cytotoxicity mediated by CD16, is dependent on PKC (23, 24), we examined whether DNAM-1-mediated cytolytic activation of NK cells requires the PKC pathway. As demonstrated in Figure 2, DX11 mAb-induced redirected cytolysis mediated by NK clones was inhibited in a dose-dependent manner by the pretreatment of NK cells with the PKC inhibitor GF109203X. In contrast, consistent with prior findings (23, 24), the PKC inhibitor did not affect anti-CD16-induced redirected cytolysis. These results indicate that the PKC pathway is involved in DNAM-1-mediated cytolytic activation of NK cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Anti-DNAM-1 mAb redirected cytolysis against murine FcR-bearing mastocytoma P815 targets mediated by a NK clone. PKC inhibitor GF109203X suppressed DX11 mAb-redirected cytolysis against P815 in a dose-dependent manner. By contrast, anti-CD16-induced redirected cytolysis was not affected.

Since PKC activation plays an important role in DNAM-1-mediated cytotoxicity, we examined the effect of PKC inhibitor GF109203X on DNAM-1 adhesion. As demonstrated in Figure 3, A and B, GF109203X specifically inhibited in a dose-dependent manner the binding of Colo-205 to the DNAM-1 transfectants. Moreover, pretreatment of the DNAM-1 transfectants with PMA accelerated the binding of the transfectants to Colo-205 (Fig. 3B). Expression of DNAM-1 on the cell surface of the transfectants was not affected by either PMA or GF109203X, as determined by flow cytometry (not shown). These results suggest that PKC is involved in DNAM-1 adhesion.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Kinetics of DNAM-1 adhesion and effect of a PKC inhibitor and PMA. BW5147 or DNAM-1+ BW5147 cells were pretreated with the PKC inhibitor GF109203X at the indicated concentration (A), at 1 µM (B), or pretreated with PMA (50 ng/ml), or medium containing 0.1% DMSO (B) for 2 h at 37°C. Cells were then assayed for binding to Colo-205 cells, as described in Materials and Methods.

DNAM-1 contains two putative PKC-binding sites at residues 293 and 329 in the cytoplasmic domain (13). To determine whether elements in the cytoplasmic domain are necessary for DNAM-1 binding, BW5147 transfectants stably expressing several DNAM-1 truncation mutants were established (designated 287, 298, 308, and 327) (Fig. 4, A and B). Although the amount of DNAM-1 glycoprotein expressed on the cell surface of these transfectants was comparable with the wild-type DNAM-1 transfectant (Figs. 1A and 4B), Colo-205 did not bind to any of these transfectants (Fig. 4C). Furthermore, pretreatment of these transfectants with PMA did not augment binding (Fig. 4C), suggesting that the amino acids responsible for PMA-induced DNAM-1 adhesion might be located in the cytoplasmic domain (Fig. 4A). Because the DNAM-1 mutant containing a stop codon at residue 327 failed to bind, we examined whether the putative PKC phosphorylation site at Ser³²⁹ might be required for adhesion. Therefore, we created a BW transfectant expressing a mutant DNAM-1 molecule with a site-directed mutation at residue 329, converting Ser³²⁹ to Phe³²⁹ (designated S-F³²⁹) (Fig. 4, A and B). The Phe³²⁹ DNAM-1 transfectant was unable to bind Colo-205, even after the pretreatment with PMA (Fig. 4C). These results suggested that Ser³²⁹ might be the residue that serves as a substrate for PKC and is responsible for DNAM-1 adhesion. To test this hypothesis, the wild-type and mutant DNAM-1 BW transfectants were labeled with [³²P]orthophosphate, stimulated with PMA, and DNAM-1 proteins were immunoprecipitated with DX11 mAb. As shown in Figure 5, the wild-type DNAM-1, but neither the deletion mutant (327) nor Phe³²⁹-DNAM-1 mutant (S-F³²⁹), was phosphorylated after stimulation with PMA. Thus, Ser³²⁹ appears to be the site of serine phosphorylation by activated PKC and this event is critical for DNAM-1 adhesion.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Establishment of BW5147 transfectants with mutant DNAM-1. A, The amino acid sequence of DNAM-1 cytoplasmic domain is shown. The several mutant cDNA-inserting stop codons at the indicated sites in the cytoplasmic domain of DNAM-1 were generated by PCR and subcloned into a retroviral vector. 287, 298, 308, and 327 are BW5147 transfectants expressing truncated DNAM-1 molecules. S-F329 is a BW5147 transfectant expressing DNAM-1 with a site-directed mutation at residue 329 (Phe329). B, The transfectants were stained with either PE-conjugated control IgG (open histograms) or PE-conjugated anti-DNAM-1 mAb DX11 (shaded histograms), and analyzed by flow cytometry. The transfectants stably express the wild-type or mutant DNAM-1 Ag at comparable levels. C, Binding of transfectants expressing mutant DNAM-1 receptors to Colo-205 cells. BW5147 cells or BW5147 transfectants expressing wild-type DNAM-1, a DNAM-1 deletion mutant (327) or the Phe329-DNAM-1 mutant (S-F329) were pretreated or not with PMA (50 ng/ml for 2 h) and then assayed for binding of Colo-205 cells for the indicated time at 37°C.

View larger version (90K): [in this window] [in a new window]   FIGURE 5. Ser329 of DNAM-1 is phosphorylated by PKC. DNAM-1+ BW5147 transfectants were metabolically labeled with [32P]orthophosphate and then stimulated or not with 50 ng/ml PMA for the indicated time. The transfectants were lysed in 1% Nonidet P-40, lysates were immunoprecipitated with control Ig or DX11 mAb, and samples were analyzed by SDS-PAGE in nonreducing sample buffer. DNAM-1 phosphorylation was observed in the PMA-stimulated transfectants expressing wild-type DNAM-1, but not in transfectants with a truncated DNAM-1 mutant (327) or the Phe329-DNAM-1 mutant (S-F329).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The consensus sequences for PKC phosphorylation sites are S or T residue flanked on one or both sides by a basic amino acid, K or R, sometimes separated by one or two "spacer" amino acids (27). DNAM-1 contains two putative PKC phosphorylation sites, T(Q)K and SRR at residues 293 and 329, respectively, in the cytoplasmic domain (Fig. 4A). We have demonstrated that mutation of the phosphorylation site at residue 329 from S to F completely inhibits DNAM-1 binding and the phosphorylation of DNAM-1 induced by PMA. These results indicate that S³²⁹ phosphorylation by PKC is required for DNAM-1 adhesion.

In many respects, DNAM-1 is similar to the hemopoietic cell-specific integrin LFA-1. For example, LFA-1 and DNAM-1 are expressed on NK and T cells, and mAbs against these molecules can partially, or totally, inhibit cell-mediated cytotoxicity (13, 28, 29). Moreover, intercellular binding of LFA-1 or DNAM-1 to cells bearing their ligands is temperature dependent and enhanced by PMA activation of PKC (5). However, in contrast to DNAM-1, the S residue in the cytoplasmic domain of the LFA-1ß subunit that is the major substrate for PKC phosphorylation is not necessary for LFA-1 adhesion (11), although the cytoplasmic domain of LFA-1ß, but not , is required for binding to ICAM-1 (12). Furthermore, the binding of LFA-1, but not DNAM-1, requires the presence of divalent cations such as Mg²⁺ and Ca²⁺ (5). It takes a period of hours for DNAM-1 to reach maximal adhesion, whereas binding of LFA-1 to its ligands is rapidly accelerated following T cell activation, with maximal binding in minutes (30, 31, 32, 33). DNAM-1 is a monomeric glycoprotein of the Ig superfamily (11); LFA-1 is a heterodimer composed of two noncovalently associated subunits of the integrin family (34). While the ligands for LFA-1, the ICAMs, are members of the Ig superfamily (25), the cellular ligand for DNAM-1 has not yet been identified.

We have presented evidence for a critical role of PKC for both DNAM-1 signaling and adhesion. PKC is activated by signals through the TCR/CD3 complex on T cells (5). Therefore adhesion mediated by DNAM-1, as well as LFA-1, may be accelerated following recognition of Ag presented by MHC on target cells by the TCR/CD3 complex on T cells. It is possible that intercellular binding between T cells and APC is mediated first by LFA-1/ICAMs adhesion within a period of minutes and then sequentially enhanced by DNAM-1/DNAM-1L adhesion to permit sustained signaling. PKC is also activated by signals through several other leukocyte surface receptors, including CD2 (5). Since essentially all CTL and NK cells express CD2, it is possible that DNAM-1 adhesion to its ligand on target cells is facilitated by a signal through CD2. Also, our studies demonstrated that specific inhibitors of PKC prevented DNAM-1-mediated signaling for cytolytic activation of NK cells, suggesting the possibility that DNAM-1 "outside-in" signaling may activate PKC and initiate DNAM-1 adhesion. This is consistent with the observation that the transfectant with wild-type DNAM-1 significantly binds Colo-205 without stimulation by PMA (Fig. 4A).

In summary, our studies have shown that PKC plays an important role for both DNAM-1-mediated signaling and adhesion. T and NK cells express various adhesion molecules including LFA-1, CD2, and DNAM-1. The role of the adhesion molecules in lymphocyte-APC interactions may be dictated by a dynamic process and influenced by signals generated through other membrane receptors. We have identified certain cell lines that specifically bind to DNAM-1 transfectants (13) and are presently pursuing these molecules. The identification and molecular characterization of DNAM-1L would contribute to understanding the structural requirements involved in DNAM-1 signaling and binding.

	Acknowledgments

 
We thank Toshio Kitamura, Joe Bolen, Chiwen Chang, Kazuko Shibuya, and Eiichi Nakayama for helpful discussion; Jim Cupp, Dixie Polakoff, and Eleni Calls for expert assistance with flow cytometry; and Debbi Liggett, Dan Gorman, Allison Helms, and Connie Huffine for oligonucleotides and DNA sequencing. DNAX Research Institute is supported by the Schering-Plough Corporation.

	Footnotes

 
¹ Present address: Department of Parasitology and Immunology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.

² Address correspondence and reprint requests to Joseph H. Phillips, Department of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304.

³ Abbreviations used in this paper: DNAM-1, DNAX accessory molecule-1; DNAM-1L, DNAM-1 ligand; PE, phycoerythrin; PKC, protein kinase C.

Received for publication November 20, 1997. Accepted for publication April 13, 1998.

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