Transmembrane adapter proteins are a class of molecules that mediate signals from an extracellular receptor to the cytoplasm of the cell. We have cloned a novel transmembrane adapter protein called KAP10, a ∼10-kDa protein that is encoded within 100 bp of the DAP12 locus on human chromosome 19. KAP10 is predominantly expressed in immune cells, including NK cells, T cells, and monocytes. We show that KAP10, unlike other transmembrane adapter proteins, binds phosphatidylinositol-3 kinase following phosphorylation of a cytoplasmic YINM motif, which results in activation of Akt. In addition, we identify KAP10 as being able to bind the adapter protein Grb2. Based on our data, we suggest that this molecule is involved in stimulation and costimulation in cells of both myeloid and lymphoid origin.

Transmembrane (TM)4 adapter molecules associate with various activating receptors in a wide variety of different cell types. This group of proteins includes the CD3 chains and CD79, which associate with T cell and B cell receptors, respectively. A further example is the Fc common γ-chain, which associates with both Fc receptors and activating Ig-like transcript (ILT) (1, 2, 3, 4). More recently, an adapter molecule called DAP12 (KARAP) has been shown to associate with MHC class I-specific killer cell receptors (5, 6, 7, 8, 9). TM adapter proteins usually exist as disulfide-linked hetero- or homodimers and share several features, including one or more immunoreceptor-based tyrosine activation motifs (ITAM) (D/ExxYxxL/I x6–8 YxxL/I) in their cytoplasmic tails and the ability to recruit src homology domain-2 (SH2)-containing signaling proteins, such as ZAP70 and Syk, following tyrosine phosphorylation.

Other TM costimulatory molecules, such as CD28, provide a second “survival” signal to cells, such as T cells, by using a single SH2-binding site to recruit further signaling molecules such as phosphatidylinositol-3 kinase (PI-3 kinase), Grb2, and Shc (10). These receptor networks provide cells with a sophisticated repertoire of signals that determine survival, activation, or differentiation.

We have identified and cloned a novel cDNA encoding a protein that is an outriding member of the TM adapter protein class. The corresponding gene is closely linked to DAP12. This novel protein has a YINM motif more reminiscent of that found in the family of costimulatory molecules. We show that this molecule is expressed in immune cells and, like CD28 and others, can bind both PI-3 kinase and Grb2 following stimulation. We call this molecule PI-3 kinase-associated protein of ∼10 kDa (KAP10).

Rabbit anti-human KAP10 was generated by immunizing with a peptide spanning the entire cytoplasmic tail of KAP10. Anti-CD4 mAb L3T4 are from PharMingen (San Diego, CA); anti-PI-3 kinase, anti-Grb2, and the phosphotyrosine mAb are from Transduction Laboratories (Lexington, KY). HRP-conjugated goat anti-mouse or -rabbit Ab were from Immunotech (Marseille, France). F(ab′)2 goat anti-rat IgG was from Jackson ImmunoResearch (West Grove, PA). Abs specific for phosphorylated Akt (at Thr308) and Akt Ab were from New England Biolabs (Beverly, MA). The following cell lines were used: Jurkat, NKL, NK92, U937, THP-1, RAJI, LCL721, 293T, YT, COS, SY5Y, SK-N-BE, IMR32, and a polyclonal NK cell line (NK9449).

A genomic clone that contained the DAP12 sequence (gb: AD000864) was analyzed using the NIX program at the Human Genome Mapping Program (HGMP) resource center (http://www.hgmp.mrc.ac.uk/) to determine the presence of other genes. Expressed sequence tag (EST) clones (IMAGE clones; 955952, 462536, and 331055) were obtained from the HGMP and sequenced using a 377 cycle sequencer (Applied Biosystems, Foster City, CA). A human multiple tissue northern blot (Clontech, Palo Alto, CA) was probed with a radiolabeled 285-bp KAP10 DNA fragment derived by PCR amplification from a KAP10 EST clone using primers 5′-ATGATCCATCTGGGTCACATC-3′ and 5′-TCAGCCCCTGCCTGGCAT-3′ according to the manufacturer’s instructions. Membranes were washed under high stringency conditions and exposed to x-ray film for 2–4 days.

A CD4-KAP10 chimeric construct encoding the extracellular and TM region of murine CD4 and the cytoplasmic region of human KAP10 was produced by PCR as previously described (11). The PCR product was cloned into the expression vector pCDNA-3 and transfected into Jurkat T cells. After 3–4 wk of selection, G418-resistant clones were expanded and maintained in medium without G418.

Peptides were synthesized by MWG Biotec (Ebersberg, Germany) (KAP10-pY71 and KAP10-Y71) and Zinsser Analytic (Frankfurt, Germany) (pY740/pY751) and were of the following sequences: KAP10-Y71- CPAQEDGKYINMPGRG, KAP10-pY71-KSPAQEDGKpYINMPGRG, and pY740/pY751-GGpYMDMSKDESVDpYVPML, where pY represents a phosphotyrosine residue. Phosphatidylinositol was purchased from Sigma (St. Louis, MO). Synthetic peptides were covalently coupled to Actigel ALD-superflow beads (Sterogene Bioseparations, Carlsbad, CA) as described by the manufacturer, at a peptide concentration of 1 mg/ml. Jurkat and NKL cells were lysed using Triton X-100 lysis buffer (1% Triton X-100, 20 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 5 mM 2-ME, and protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN)), the nuclei were removed by centrifugation, and cell lysates were precleared with protein A-Sepharose beads. Cell lysates were incubated with 20 ml (20 mg of peptide) of peptide coupled to Actigel.

PI-3 kinase assays were conducted as previously described (12). Extracted phospholipids were analyzed by TLC. TLC plates were exposed to a phosphor screen and images analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Cells (2 × 107) were washed in PBS and treated with pervanadate for 10 min at room temperature. Cell lysates were incubated for 2 h at 4°C with 2 μg of anti-CD4 mAb and then incubated with protein G-Sepharose beads for 1.5 h. After precipitation, the beads were washed five times with lysis buffer before elution of the proteins by boiling for 5 min in SDS-PAGE sample buffer. Samples were electrophoresed in 12% polyacrylamide gels and proteins were transferred to polyvinylidene difluoride (PVDF)-membranes (Hybond-P, Amersham Laboratories, Amersham, U.K.) by semidry blotting. Membranes were blocked in 4% nonfat milk in PBS containing 0.1% Tween 20 for 1 h. PI-3 kinase, Grb2, or tyrosine phosphorylated proteins were detected by appropriate Abs followed by HRP secondary reagents. Bound HRP-conjugated goat anti-mouse or -rabbit Ab was visualized using enhanced chemiluminescence (ECL, Amersham).

Cells were incubated for 1 h in the presence 1 μM PI-3 kinase inhibitor wortmannin (Calbiochem, La Jolla, CA) or in medium. Thereafter, cells were stimulated with anti-CD4 mAb (L3T4) and cross-linker (F(ab′)2 goat anti-rat IgG for the indicated time periods. Cells were then lysed in sample buffer and boiled for 5 min. Following SDS-PAGE and transfer to PVDF membrane (Hybond-P, Amersham Laboratories), phosphorylated Akt was detected with a phospho-Akt Ab (New England Biolabs) followed by an HRP-conjugated goat anti-rabbit Ab (Immunotech). Membranes were stripped at 55°C for 1 h and reprobed with anti-Akt Ab.

There are many instances in the mammalian genome of linked genes with related functions, such as the MHC and the CD3 gene complex (13, 14). We searched in the vicinity of the human DAP12 gene for related sequences. A novel 2.0-kb gene was identified on a genomic clone containing the DAP12 gene. We obtained corresponding EST clones. Comparison of the composite cDNA with the human genomic sequence revealed that the gene, which we termed KAP10, comprises four exons and is located in a tail-to-tail orientation within 100-bp of DAP12. As shown in Fig. 1, within the 504-bp cDNA clone is an open reading frame (ORF) of 282 bp, encoding a putative type I membrane protein of 94 amino acids with a molecular mass of ∼9.5 kDa. The amino acid sequence of KAP10 included a putative 19 amino acid signal peptide. The molecular mass of the mature protein is 7.5 kDa. The predicted extracellular domain spans 25 amino acids and contains two cysteine residues proximal to the TM. An aspartate residue is found in the middle of the TM domain, a feature similar to DAP12. Comparison with either CTLA-4 or CD28 reveals homology surrounding the cytoplasmic tyrosine residue and provides some insight as to the function of KAP10 (Fig. 1 B). The cytoplasmic tail contains a tyrosine-containing motif (YINM) corresponding to a putative PI-3 kinase SH2 domain-binding motif (pYxxM) (15), a Grb2 site (pYxN), and an Shc site (pY-Y[E/I]x[I/L/M]) (16).

FIGURE 1.

Nucleotide and deduced amino acid sequences of human KAP10. Nucleotide sequence was compiled from EST clones and RT-PCR products. A, The deduced amino acid sequence is shown below the nucleotide sequence. The putative signal peptide is single underlined, the predicted TM domain is dotted underlined, and the PI-3 kinase binding domain is double underlined. The stop codon is indicated by an asterisk, and the untranscribed sequence is in lower case. The KAP10 sequence is deposited under Genbank accession number AF172929. B, Comparison of the amino acid sequence surrounding the tyrosine motifs from the cytoplasmic domains of KAP10 with CD28 and CTLA-4. The tyrosine motifs are in bold.

FIGURE 1.

Nucleotide and deduced amino acid sequences of human KAP10. Nucleotide sequence was compiled from EST clones and RT-PCR products. A, The deduced amino acid sequence is shown below the nucleotide sequence. The putative signal peptide is single underlined, the predicted TM domain is dotted underlined, and the PI-3 kinase binding domain is double underlined. The stop codon is indicated by an asterisk, and the untranscribed sequence is in lower case. The KAP10 sequence is deposited under Genbank accession number AF172929. B, Comparison of the amino acid sequence surrounding the tyrosine motifs from the cytoplasmic domains of KAP10 with CD28 and CTLA-4. The tyrosine motifs are in bold.

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Northern blot analysis using a KAP10-specific probe identified a ∼500-bp transcript (Fig. 2,A). KAP10 was shown to be expressed in peripheral blood, spleen, and thymus while KAP10 transcripts were undetectable in prostate, testis, ovary, small intestine, or colon. RT-PCR revealed expression in monocyte (U937, THP1), T (Jurkat, H9), B (RAJI, LCL721) and NK (NK92, NKL) cell lines and in decidual NK and macrophage cDNA libraries but not the fibroblast cell line 293T, nor the neural cell lines IMR32, SK-N-BE, and SY5Y. This expression pattern shows that KAP10 transcripts are predominantly expressed in cells of a hemopoietic lineage. KAP10 protein was detected by Western blotting. Under reducing conditions, KAP10 ran at ∼10 kDa and could be identified in extracts of primary NK cells, CD8 T cells, and myeloid cell lines, and to a lesser extent in Jurkat T cells (Fig. 2,B). Although KAP10 was also observed in some EBV-transformed B cell lines (e.g., C1R), its expression in normal B cells has yet to be determined. Under nonreducing conditions, KAP10 migrated as a 20-kDa protein in CD8 T cells (Fig. 2 C) and in KAP10-transfected COS cells (data not shown), suggesting that KAP10 may be present as a homodimer or a heterodimer, possibly via disulfide linkage between either of the cysteines in the extracellular domain.

FIGURE 2.

Expression of KAP10 in different tissues and cell lines. A, KAP10-specific probe was hybridized to a multiple tissue Northern blot and then washed under high stringency conditions. The blot was exposed for 2 days. The mRNA are as follows: lane 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; 8, peripheral blood leukocyte. The same blot was probed with a control β-actin probe (lower panel) to check for similar loading of mRNA in each lane. B, A polyclonal antiserum was used against the cytoplasmic tail of KAP10 for Western blot against the cell lines COS, NK9449, Jurkat T cells, and YT NK cells. C, CD8+ T cells were screened with the KAP10 antiserum under reducing (+) and nonreducing conditions (−) as described in Materials and Methods. The molecular masses are given at the side in kDa.

FIGURE 2.

Expression of KAP10 in different tissues and cell lines. A, KAP10-specific probe was hybridized to a multiple tissue Northern blot and then washed under high stringency conditions. The blot was exposed for 2 days. The mRNA are as follows: lane 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; 8, peripheral blood leukocyte. The same blot was probed with a control β-actin probe (lower panel) to check for similar loading of mRNA in each lane. B, A polyclonal antiserum was used against the cytoplasmic tail of KAP10 for Western blot against the cell lines COS, NK9449, Jurkat T cells, and YT NK cells. C, CD8+ T cells were screened with the KAP10 antiserum under reducing (+) and nonreducing conditions (−) as described in Materials and Methods. The molecular masses are given at the side in kDa.

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The YINM motif in the cytoplasmic domain of KAP10 is reminiscent of the PI-3 kinase binding motifs found in CD28 and CTLA-4. We showed that the KAP10 sequence is also a binding site for PI-3 kinase. Initially, synthetic peptides corresponding to this motif were used. Peptides KAP10-pY71 and KAP10-Y71, representing the phosphorylated and nonphosphorylated versions of the amino acid sequence, were coupled to an affinity matrix Actigel and incubated with whole cell extracts of Jurkat and NKL cells. PI-3 kinase activity was associated specifically with the tyrosine-phosphorylated KAP10 peptide and not its unphosphorylated counterpart (Fig. 3,A). A peptide corresponding to the PI-3 kinase binding site upon the platelet-derived growth factor receptor (PDGFR-pY740/pY751), precipitated a relatively greater quantity of PI-3 kinase activity, most likely due to differences in the activation potential of this peptide, sicne this site consists of two phosphotyrosines capable of dimerizing PI-3 kinase (17), as compared with the single phosphotyrosine site present in the KAP10 sequence. These results demonstrate that phosphorylated KAP10 can bind native PI-3 kinase from NKL and Jurkat T cells. To confirm that the endogenous KAP10 protein also associated with PI-3 kinase, we used an anti-KAP10 antiserum for immunoprecipitation experiments from pervanadate-treated NKL cells (Fig. 3 B). As predicted by our in vitro peptide binding data, native KAP10 was found to have associated PI-3 kinase activity.

FIGURE 3.

Association of PI-3 kinase with KAP10. A tyrosine phosphopeptide derived from the intracellular domain of the KAP10 adapter protein was used for lipid kinase assays on (A) precipitates from whole cell detergent extracts of NKL (open bars) or Jurkat (filled bars) cells incubated with the Actigel-coupled peptides pY740/pY751 (positive control), KAP10-pY71, or KAP10-Y71, or with Actigel alone. B, Anti-KAP10 peptide antisera (open bar) or the prebleed (filled bar) was used to immunoprecipitate KAP10 from pervanadate-treated NKL cells and was tested for associated PI-3 kinase activity.

FIGURE 3.

Association of PI-3 kinase with KAP10. A tyrosine phosphopeptide derived from the intracellular domain of the KAP10 adapter protein was used for lipid kinase assays on (A) precipitates from whole cell detergent extracts of NKL (open bars) or Jurkat (filled bars) cells incubated with the Actigel-coupled peptides pY740/pY751 (positive control), KAP10-pY71, or KAP10-Y71, or with Actigel alone. B, Anti-KAP10 peptide antisera (open bar) or the prebleed (filled bar) was used to immunoprecipitate KAP10 from pervanadate-treated NKL cells and was tested for associated PI-3 kinase activity.

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To investigate KAP10-mediated signaling, we generated murine CD4-human KAP10 chimeras. The chimera was expressed in Jurkat T cells, and expression was determined by FACS using an anti-murine CD4 mAb (Fig. 4,A). Cells were then treated with the tyrosine phosphatase inhibitor pervanadate to induce tyrosine phosphorylation. Immunoprecipitation of CD4-KAP10 followed by immunoblotting with a phosphotyrosine-specific mAb showed that KAP10 was phosphorylated following pervanadate treatment (Fig. 4,B). Additionally, probing immunoprecipitates with appropriate mAbs showed a constitutive association of both PI-3 kinase and Grb2 with KAP10, which was significantly increased upon phosphorylation of KAP10 (Fig. 4, C and D, respectively). Thus, KAP10 contains a binding motif for both Grb2 and PI-3 kinase and recruits both these proteins in a phosphorylation-dependent manner.

FIGURE 4.

Phosphorylated KAP10 recruits PI-3 kinase and Grb2 in vivo. Mouse CD4-human KAP10 chimera was transfected into Jurkat T cells. Jurkat CD4-KAP10 cells (open profile) were tested for cell surface expression by FACS analysis with an anti-murine CD4 Ab as compared with Jurkat T cells (filled profile) (A). CD4-KAP10 was precipitated from transfected cells either stimulated with pervanadate (+) or left untreated (−). Immunoprecipitates were analyzed by Western blot using either an anti-phosphotyrosine mAb (B), an anti-PI-3 kinase mAb (C), or a Grb2 mAb (D). Igh and Igl refer to Ig heavy and light chains, respectively.

FIGURE 4.

Phosphorylated KAP10 recruits PI-3 kinase and Grb2 in vivo. Mouse CD4-human KAP10 chimera was transfected into Jurkat T cells. Jurkat CD4-KAP10 cells (open profile) were tested for cell surface expression by FACS analysis with an anti-murine CD4 Ab as compared with Jurkat T cells (filled profile) (A). CD4-KAP10 was precipitated from transfected cells either stimulated with pervanadate (+) or left untreated (−). Immunoprecipitates were analyzed by Western blot using either an anti-phosphotyrosine mAb (B), an anti-PI-3 kinase mAb (C), or a Grb2 mAb (D). Igh and Igl refer to Ig heavy and light chains, respectively.

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It has been reported that CD28 stimulation triggers recruitment of PI-3 kinase, leading to Akt (PKB/RAC; protein kinase B/related to A and C protein kinases) activation and ultimately to cell survival (reviewed in Ref. 18). Since KAP10 also recruits PI-3 kinase, we tested whether stimulation of KAP10 leads to a similar activation of Akt. The phosphorylation status of Akt was assessed using a phospho-Akt-specific Ab. We found that KAP10 cross-linking led to phosphorylation of Akt (Fig. 5,A). Akt phosphorylation was dependent upon the activity of PI-3 kinase, since it was abolished in the presence of the PI-3 kinase inhibitor wortmannin. (Fig. 5 B). By influencing the activation status of Akt, KAP10 may be involved in transducing an important cell survival signal.

FIGURE 5.

KAP10 cross-linking leads to phosphorylation of Akt through PI-3 kinase activation. A, CD4-KAP10 Jurkat cells were treated with an anti-CD4 Ab and a cross-linking Ab for the time periods specified. Cell lysates were analyzed for phosphorylated Akt by Western blot analysis using a phospho-Akt-specific Ab. The amount of Akt was confirmed by reblotting with an anti-Akt Ab (Akt). B, Cells were stimulated by cross-linking the CD4-KAP10 for different time periods in the presence (+ wort) or absence (− wort) of the PI-3 kinase inhibitor wortmannin. Phospho-Akt and Akt were detected as above.

FIGURE 5.

KAP10 cross-linking leads to phosphorylation of Akt through PI-3 kinase activation. A, CD4-KAP10 Jurkat cells were treated with an anti-CD4 Ab and a cross-linking Ab for the time periods specified. Cell lysates were analyzed for phosphorylated Akt by Western blot analysis using a phospho-Akt-specific Ab. The amount of Akt was confirmed by reblotting with an anti-Akt Ab (Akt). B, Cells were stimulated by cross-linking the CD4-KAP10 for different time periods in the presence (+ wort) or absence (− wort) of the PI-3 kinase inhibitor wortmannin. Phospho-Akt and Akt were detected as above.

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Like other TM adapter proteins such as CD3 and DAP12, KAP10 has a short extracellular domain and a charged residue in the TM. It is therefore likely that KAP10 also associates with a partner bearing a charged residue in its TM domain, such as an NK receptor. Now that we have a clearer understanding of the intracellular interactions of this unique TM adapter protein, it will be of great interest to determine its binding partners in different cell lineages.

Note.

KAP10 was recently described as DAP10 by Wu et al., who showed that one of its partner chains is the C-type lectin NKG2D (19).

We thank Michaela Torkar, Nick Holmes, Derek McCusker, and Rachel Allen for helpful discussions. We also thank Philippe Bastiaens for the use of his laboratory, Meredith Layton for the generous donation of recombinant PI-3 kinase, and Damian Crowther for SY5Y cells. Jacqueline Samaridis and Lena Angman are gratefully acknowledged for their excellent technical support.

1

J.D. is a recipient of a postdoctoral fellowship from the Danish Medical Research Counsel. This work was supported by the Medical Research Council and The Wellcome Trust. The Basel Institute of Immunology was founded and is supported by Hoffman-La Roche, Ltd., Basel, Switzerland.

4

Abbreviations used in this paper: TM, transmembrane; PI-3 kinase, phosphatidylinositol-3 kinase; DAP12, DNAX-activating protein of 12 kDa; SH2, src homology domain-2; KAP10, PI-3 kinase-associated protein of ∼10 kDa; EST, expressed sequence tag; wort, wortmannin.

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