INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Engagement of the B-cell antigen receptor (BCR) and the T-cell antigen receptor (TCR) initiates multiple intracellular signals that can lead to cellular proliferation and the acquisition of complex effector functions. Analysis of sequence elements responsible for the signaling properties of the transducing subunits of BCR and TCR has led to the identification of the immunoreceptor tyrosine-based activation motif (ITAM) (3, 8, 36, 37). This motif consists of six conserved amino acid residues spaced precisely over an ~26-amino acid sequence, (D/E)X₇(D/E)X₂YX₂LX₇YX₂L/I, where X is any amino acid. The ITAM is present in a number of cellular signal-transducing molecules, such as TCR-, immunoglobulin alpha (Ig), Ig, CD3, CD3, FcRI, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, and simian immunodeficiency virus PBj14 Nef (2, 3, 7, 8, 22, 23). It has been well documented that this motif is necessary and sufficient for the coupling of extracellular signals to intracellular signaling molecules. Upon stimulation, the tyrosine residues within the ITAMs become phosphorylated, permitting the binding of SH2 domain-containing proteins. Subsequent signaling molecules are recruited to these associated proteins via SH2 or other modular interaction domains (3, 8, 33, 37).

The association of ITAM-containing receptors with tyrosine kinases has been analyzed in considerable detail (32). Immediately downstream of the BCR and the TCR in the signaling pathway are the receptor-associated protein tyrosine kinases (PTKs) (3, 36, 37). Two families of PTKs have been shown to be involved in BCR and TCR signaling. Lyn, Fyn, Blk, and Lck are members of Src family, while Zap70 and Syk make up another PTK family (3, 36, 37). A primary role of the Src family kinases is to phosphorylate two tyrosine residues within the ITAMs of BCR and TCR. Subsequently, Syk and Zap70 are recruited to activated receptors, which in turn lead to the induction of cellular tyrosine phosphorylation, the elevation of intracellular calcium, the activation of lipid-dependent kinases, and the activation of Ras and its downstream kinase cascade (3, 36, 37). The cross-linking of chimeric molecules composed of the extracellular and transmembrane domains of the CD4, CD8, or CD16 molecule and a single copy of the ITAM motif has been shown to be sufficient to elicit early or late signal-transducing events (2, 12, 15, 17). Thus, ITAMs function as a scaffold to recruit and organize effector molecules upon receptor ligation.

DNA sequences of a novel member of the herpesvirus group, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8, have been widely identified in Karposi's sarcoma tumors from human immunodeficiency virus-positive and -negative patients (4, 5, 21). KSHV has also been identified in body cavity-based lymphoma and some forms of Castleman's disease (4, 5, 29). The genomic sequence shows indicates KSHV to be a gammaherpesvirus that is closely related to herpesvirus saimiri (HVS) (26, 30) and the recently isolated rhesus monkey rhadinovirus (6). DNA sequence analysis of the entire 140.5 kbp of the KSHV genome revealed a number of cellular homologs which could possibly contribute to the pathogenesis associated with this virus (26, 30). These include a virus-encoded interleukin-6 (IL-6) (24, 27, 28), MIP1-/ chemokines (14, 24, 28), a Bcl-2 homolog (31), a virus-encoded interferon regulatory factor (9, 19, 38), v-cyclin (10, 20), IL-8 receptor (1), FLICE-inhibitory protein (35), and an N-CAM homolog.

At a position equivalent to the STP (saimiri transformation protein) of HVS, KSHV contains a distinct open reading frame called K1 (18). Although KSHV and HVS are related members of the rhadinovirus subgroup of gammaherpesviruses, K1 and STP exhibit no similarity in amino acid sequence or in organization of structural motif. The K1 protein is predicted to have a signal peptide sequence at the amino terminus, an extracellular domain, a transmembrane domain, and a short cytoplasmic tail at the carboxyl terminus (16, 18, 30). The predicted extracellular domain of the K1 protein apparently influences the strength or degree of disulfide-linked oligomerization (18). Expression of the K1 gene in rodent fibroblasts produced morphologic changes and focus formation indicative of transformation. A recombinant herpesvirus in which the STP oncogene of HVS was replaced with the K1 gene immortalized primary T lymphocytes to IL-2-independent growth and induced lymphoma in common marmosets (18). These results demonstrated unambiguously the oncogenic potential of the KSHV K1 gene.

In this report, we show that the cytoplasmic region of K1 contains significant homology with the ITAM of cellular signal-transducing molecules. To demonstrate the potential signal-transducing activity of the cytoplasmic region of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8 polypeptide was replaced with that of the KSHV K1. Expression of this chimera induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. These results demonstrate that the cytoplasmic ITAM sequence of the KSHV transforming protein K1 can transduce signals to elicit cellular activation events.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cell culture and transfection. BJAB cells were grown in RPMI medium supplemented with 10% fetal calf serum (FCS). COS-1 cells were grown in Dulbecco modified Eagle medium supplemented with 10% FCS. A DEAE-dextran transfection procedure was used for transient expression in COS-1 cells. The pcDNA3-CD8 chimeric constructs (20 µg) were introduced into BJAB cells by electroporation at 250 V and 960 µF in serum-free DME medium. After a 48-h incubation, the cells were cultured with selection medium containing 2 mg of neomycin per ml for the next 5 weeks.

Plasmid constructions. DNA containing the EcoRI-BglII fragment of the K1 gene encoding amino acids 251 to 289 was amplified by PCR and was fused in frame to the human CD8 containing the deletion of its carboxyl terminus (CD8) in pSP72 vector. For stable expression, the KpnI-BglII DNA fragment containing the CD8-K1 chimera was cloned into the KpnI-BamHI site of pcDNA3 (Invitrogen, San Diego, Calif.). All mutations in the K1 gene were generated with PCR by using oligonucleotide-directed mutagenesis (7). The amplified DNA fragments containing mutations in K1 were purified and cloned into pSP72 vector. Each K1 mutant was completely sequenced to verify the presence of the mutation and the absence of any other changes. After confirmation of the DNA sequence, DNA containing the desired K1 mutation was recloned into pFJ vector or pcDNA3 vector containing CD8 for gene expression. In some cases, the CD8-K1 chimeric gene tagged with an AU-1 epitope at its carboxyl terminus was used for expression. The Syk expression plasmid was kindly provided by A. Veillette.

Immunoprecipitation and immunoblotting. COS-1 cells at 80 to 90% confluence in a 25-cm² dish were rinsed three times with phosphate-buffered saline, washed once with labeling medium (minimum essential medium minus methionine and cysteine plus 10% dialyzed FCS), and then incubated with 2 ml of the same medium containing 200 µCi of [³⁵S]methionine and [³⁵S]cysteine (New England Nuclear, Boston, Mass.) for 7 h. Cells were incubated in labeling medium for 30 min prior to addition of the radioisotopes. For immunoprecipitation, cells were harvested and lysed with lysis buffer (0.15 M NaCl, 0.5% Nonidet P-40, 50 mM HEPES buffer [pH 8.0]) containing 1 mM Na₂VO₃, 1 mM NaF, and protease inhibitors (leupeptin, aprotinin, phenylmethylsulfonyl fluoride and bestatin). Immunoprecipitated proteins were detected by autoradiography. For protein immunoblots, polypeptides in cell lysates corresponding to 10⁵ cells were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane filters. Immunoblot detection was performed with a 1:1,000 or 1:3,000 dilution of primary antibody used for the Amersham (Chicago, Ill.) enhanced chemiluminescence system.

FACS analysis. For fluorescence-activated cell sorting (FACS) analysis, 5 × 10⁵ cells were washed with RPMI medium containing 10% FCS and incubated with fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated monoclonal antibodies for 30 min at 4°C. After washing, each sample was fixed with 1% formalin solution, and FACS analysis was performed with a FACSscan (Becton Dickinson Co., Mountainview, Calif.). For cell sorting, 2 × 10⁷ cells were stained with fluorescein isothiocyanate-conjugated CD8 antibody 51.1 for 30 min at 4°C. Stained cells were sorted based on CD8 surface expression by a FACSVantage (Becton Dickinson). After sorting, cells were washed twice with phosphate-buffered saline and cultured with RPMI-10% FCS. CD8 antibody 51.1 used for FACS analysis and antibody OKT8 used for stimulation were obtained from the American Type Culture Collection.

Antibody stimulation. A total of 10⁷ cells were incubated with 10 µg of anti-CD8 antibody OKT8 at 37°C for the indicated time. After stimulation, cells were immediately frozen and lysed with cold lysis buffer containing 1 mM Na₂VO₃, 1 mM NaF, and protease inhibitors (leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and bestatin). Precleared cell lysates were used for immunoblotting or for immunoprecipitation.

Calcium mobilization analysis. A total of 2 × 10⁶ cells were loaded with 1 µM indo-1 in 2 ml of RPMI complete medium for 20 min at 37°C. The protocol has been described in detail previously (23). Baseline calcium levels were established for 1 min prior to addition of the antibody. Cells were stimulated with 10 µg of mouse anti-CD8 antibody OKT8 or mouse anti-human IgM antibody, and data were collected for 4 min. Baseline absolute intracellular calcium levels were determined by using ionophore and EGTA. Data were collected and analyzed on a FACSVantage (Becton Dickinson).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

An ITAM-like sequence in the cytoplasmic region of K1. A number of cellular lymphocyte receptors contain a common sequence motif in their cytoplasmic tails termed the ITAM (3). The KSHV K1 is predicted to have a lymphocyte receptor-type structure consisting of an extracellular immunoglobulin region, a transmembrane region, and a cytoplasmic region (18). Close inspection revealed that the cytoplasmic region of K1 contains a sequence with significant homology with ITAMs (Fig. 1A). More precisely, the cytoplasmic region of K1 contains negatively charged amino acids at conserved positions, the first YXXL motif, the seven amino acids necessary for precise spacing, and a second tyrosine-containing sequence, YXXP (Fig. 1A). Unlike other ITAMs, K1 contains a proline residue at the position of a leucine in the second YXXL motif (Fig. 1A). Proline is a suitable amino acid at this position to serve as an SH2 binding domain (33). Despite dramatic variation in K1 sequences from different sources, the putative ITAM in the cytoplasmic region is completely conserved in more than 20 K1 sequences that we have determined (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Sequence comparison of the cytoplasmic region of K1 with ITAMs and expression of CD8-K1 chimeras. (A) Sequence comparison of the cytoplasmic region of K1 with ITAMs. Boxes indicate conserved amino acids. h, human; m, mouse; BLV, bovine leukemia virus; SIV, simian immunodeficiency virus. (B) Expression of CD8-K1 chimeras in COS-1 cells. COS-1 cells were transfected with pFJ expression vector containing no insert DNA (), CD8 (), CD8-K1-C (K1-C), CD8-D1 (D1), CD8-D2 (D2), CD8-YY/FF (YY/FF), CD8-Y282F (Y282F), CD8-TYF (TYF), CD8-D3 (D3), or CD8-D4 (D4). After labeling with [35S]methionine and [35S]cysteine, lysates were used for immunoprecipitation with an anti-CD8 antibody. Locations of CD8-K1 chimeric proteins are indicated by dots; sizes are indicated in kilodaltons.

Construction of a CD8 chimera with the cytoplasmic region of K1. To determine whether the cytoplasmic region of K1 has a functional ITAM, we analyzed the signaling capacity of the cytoplasmic tail independent of the extracellular and transmembrane regions of K1. Antibody cross-linking of chimeric molecules composed of the extracellular and transmembrane domains of the CD8 molecule and a single copy of the ITAM motif has been shown to be sufficient to elicit early or late signal-transducing events (2). We constructed a chimeric protein in which 27 amino acids of the cytoplasmic tail of human CD8 protein were replaced with 38 amino acids of the cytoplasmic tail of K1 (CD8-K1-C [Fig. 2]). Also, CD8, which contains a deletion of its cytoplasmic region, was used as a control (Fig. 2). Since mutations at the tyrosine residues of the ITAM sequences of cellular receptors abrogate their signal-transducing capacity (3, 8), we also introduced mutations at the conserved tyrosine residues of the putative ITAM of K1. A series of tyrosine-to-phenylalanine mutations was generated as follows: CD8-YY/FF was mutated at positions 271 and 272 of tyrosine to phenylalanines, CD8-Y₂₈₂F was mutated at position 282 of tyrosine to phenylalanine, and CD8-TYF was mutated at positions 271, 272, and 282 of tyrosine to phenylalanines (Fig. 2). In addition, we introduced deletion mutations into the cytoplasmic region of K1, resulting in constructs CD8-D1, CD8-D2, CD8-D3, and CD8-D4 (Fig. 2). CD8-D1 contains the negatively charged conserved region with a proximal YXXL motif, CD8-D2 contains only the negatively charged conserved region, CD8-D3 contains both YXXL/P motifs without the negatively charged conserved region, and CD8-D4 contains the distal YXXP motif.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Summary of mutational analysis of CD8-K1 chimeras. Extracellular and transmembrane domains (positions 1 to 196) of CD8 are indicated by open and black boxes, respectively; the cytoplasmic region (251 to 289) of K1 is indicated by the dotted box. Scoring of activity: ++, strong; +, weak; +/, very weak; , none.

Construction of BJAB cell lines expressing CD8-K1 chimeras. To assess the signal-transducing activity of CD8-K1 chimeras, BJAB cells (KSHV and EBV negative) were used to establish stable lines expressing the CD8-K1 chimeric genes. The CD8 and CD8-K1 chimeric genes were cloned into the expression vector pcDNA3. After electroporation of the expression vector into BJAB cells, cell lines were selected by growth in medium containing 2 mg of neomycin per ml for 5 weeks. Since CD8 is not expressed on the surface of BJAB cells, neomycin-resistant cells were sorted by FACS analysis based on the surface expression of CD8. Comparable levels of CD8 surface expression of FACS-sorted cells were detected in most of the cells expressing CD8-K1 chimeras with the exception of CD8 cells (Fig. 3). The reduced level of CD8 surface expression in CD8 cells was likely caused by the absence of its cytoplasmic region. Subsequently, we measured the CD8 surface expression on FACS-sorted cells after they had been in culture more than a month. CD8 surface expression remained stable in most cell lines with the exception of CD8-K1-C, in which the CD8 surface expression declined over the culture period (data not shown). Based on these results, the CD8 chimera cells which had been in culture less than 1 month were exclusively used for subsequent experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Flow cytometric analysis of surface CD8 expression on BJAB cell lines. Live cells were stained for surface expression of CD8 as described in Materials and Methods. Two hundred thousand events were collected on a FACScan flow cytometer. For control, the dark-shaded histogram of each cell line is overlaid with the histogram of the BJAB cells on the solid line. (A) CD8 (mean of the gated cells for the CD8 surface expression [M], 365); B, CD8-K1-C (M = 2,289); C, CD8-D1 (M = 4,393); D, CD8-D2 (M = 4,576); E, CD8-D3 (M = 1,104); F, CD8-D4 (M = 1,730); G, CD8-YY/FF (M = 3,323); H, CD8-Y282F (M = 3,364); I, CD8-TYF (M = 1,206).

Intracellular calcium mobilization. As a control, we examined the ability of BCR to transduce signals in BJAB cells expressing CD8 or the CD8-K1-C chimera. The surface expression of IgM by FACS analysis showed equivalent levels of IgM surface expression on BJAB cells expressing CD8 or CD8-K1-C (Fig. 4A). Cells were treated with anti-IgM antibody and analyzed with flow cytometry to monitor the intracellular free-calcium levels. Stimulation with anti-IgM antibody induced a rapid increase in intracellular calcium concentration in both cell lines at comparable levels (Fig. 4B). Seven different cell lines expressing mutant forms of CD8-K1 chimeras were analyzed in this same assay. All cell lines expressing mutant forms of CD8-K1 chimeras were able to elicit levels of intracellular free-calcium mobilization similar to those elicited by cells expressing CD8 or CD8-K1-C (data not shown). This result suggests that the BJAB cell lines expressing CD8 or CD8-K1 chimeras were similarly capable of inducing intracellular signals through the BCR. To determine the ability of the putative ITAM of K1 to elicit an increase in cytoplasmic free calcium, we treated BJAB cells expressing CD8 or the CD8-K1-C chimera with anti-CD8 antibody OKT8, and monitored the intracellular free-calcium levels by flow cytometry in three independent experiments. While control CD8 cells showed no change in intracellular free-calcium concentration upon anti-CD8 stimulation, CD8-K1-C cells exhibited a prolonged increase in intracellular calcium concentration immediately after anti-CD8 treatment (Fig. 5). Seven different cell lines expressing mutant forms of CD8-K1 chimeras were analyzed in this same assay. None of the cell lines expressing mutant forms of CD8-K1 chimeras were able to elicit an increase of intracellular free-calcium concentration (Fig. 5). Thus, the putative ITAM of K1 is capable of transducing a signal to elicit intracellular calcium mobilization, and an intact ITAM sequence of K1 is required for this activity.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Surface expression of IgM and induction of intracellular free-calcium concentration. (A) Level of IgM surface expression. BJAB expressing CD8 or CD8-K1-C cells were examined for surface expression of IgM on by FACS analysis. (B) Induction of intracellular free-calcium concentration after cross-linking with anti-IgM antibody. Calcium mobilization was monitored over time by changes in the ratio of violet to blue (405 to 485 nm) fluorescence of cells loaded with the calcium sensitive dye indo-1 and analyzed by flow cytometry. Data are presented as a histogram of the number of cells with a particular ratio of blue fluorescence (y axis) over the time (seconds) after anti-IgM cross-linking (x axis). Data were reproduced in three independent experiments. The break in the graph on the left indicates the interval during addition of antibody. Numbers inside the boxes indicate the percentages of cells which responded to stimulation.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Induction of intracellular free calcium after stimulation with anti-CD8 antibody (see the legend to Fig. 4 for details). Data are presented as a histogram of the number of cells with a particular ratio of blue fluorescence (y axis) over the time (seconds) after anti-CD8 cross-linking (x axis). Data were reproduced in three independent experiments. The break in the graph on the left indicates the interval during addition of antibody. (A) CD8; (B) CD8-K1-C; (C) CD8-YY/FF; (D) CD8-Y282F; (E) CD8-TYF; (F) CD8-D1; (G) CD8-D2; (H) CD8-D3; (I) CD8-D4.

Induction of cellular tyrosine phosphorylation upon stimulation. The biochemical event subsequent to TCR or BCR stimulation is the induction of tyrosine phosphorylation of a number of cellular proteins (3, 36). We examined the effects of CD8-K1 chimera expression on cellular tyrosine phosphorylation upon stimulation with anti-CD8 antibody. BJAB cells expressing either the CD8 or the CD8-K1-C chimera were stimulated with an anti-CD8 antibody, and the course of tyrosine phosphorylation induction was observed by immunoblot assay with an antiphosphotyrosine antibody (Fig. 6A). Stimulation with an anti-CD8 antibody did not induce cellular tyrosine phosphorylation in BJAB cells expressing CD8 (Fig. 6A). Furthermore, decreased tyrosine phosphorylation of a 69-kDa protein was detected in these cells (Fig. 6A). In contrast, stimulation with an anti-CD8 antibody rapidly induced tyrosine phosphorylation of a number of proteins in BJAB cells expressing CD8-K1-C within 1 min of stimulation (Fig. 6A). Proteins of 53, 55, 70, 120, and 160 kDa displayed an increase in tyrosine phosphorylation after stimulation in BJAB cells expressing CD8-K1-C.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Induction of cellular tyrosine phosphorylation after stimulation with an anti-CD8 antibody. (A) Induction of tyrosine phosphorylation by antibody stimulation over incubation time. A total of 5 × 106 cells were incubated with anti-CD8 antibody OKT8 at 37°C for the indicated time and lysed with lysis buffer. Precleared cell lysates were used for immunoblotting with an antiphosphotyrosine antibody. (B) Induction of cellular tyrosine phosphorylation by CD8-K1 mutants. A total of 5 × 106 cells were incubated without () or with (+) anti-CD8 antibody at 37°C for 1 min and lysed with lysis buffer. Precleared cell lysates were used for immunoblotting with an antiphosphotyrosine antibody. Lanes: 1 and 2, CD8; 3 and 4, CD8-K1-C; 5 and 6, CD8-D1; 7 and 8, CD8-D2; 9 and 10, CD8-D3; 11 and 12, CD8-D4; 13 and 14, CD8-YY/FF; 15 and 16, CD8-Y282F; 17 and 18, CD8-TYF. Arrows indicate proteins with increased tyrosine phosphorylation; sizes are indicated in kilodaltons.

Tyrosine phosphorylation of Syk, Cbl, Vav, and p85 of PI3-kinase. Antibody stimulation induced the tyrosine phosphorylation of 53-, 55-, 70-, 120-, and 160-kDa proteins in BJAB cells expressing CD8-K1 chimeras (Fig. 6). We attempted to identify these tyrosine-phosphorylated cellular proteins in stimulated cells. Cellular Blk, Syk, p85 of phosphatidylinositol 3-kinase (PI3-kinase), Vav, and Cbl, which are similar in molecular mass to these proteins, have been shown to be tyrosine phosphorylated upon BCR or TCR stimulation (3, 8, 37). To examine the tyrosine phosphorylation of these cellular proteins, a specific antibody was used for immunoprecipitation from cells with and without antibody stimulation. Immune complexes were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and reacted with an antiphosphotyrosine antibody. Increased tyrosine phosphorylation of Syk was detected in BJAB cells expressing CD8-K1-C after stimulation, while it was not detected in other cell lines (Fig. 7). Tyrosine phosphorylation of Cbl was dramatically increased in BJAB cells expressing CD8-K1-C, CD8-D1, CD8-YY/FF, or CD8-Y₂₈₂F after stimulation, although the levels of tyrosine phosphorylation varied for each cell line (Fig. 7). Also, the tyrosine phosphorylation of p85 of PI3-kinase was strongly increased in BJAB cells expressing CD8-K1-C, CD8-YY/FF, or CD8-Y₂₈₂F (Fig. 7). In contrast, the tyrosine phosphorylation of Vav and Blk was not affected in these cells after stimulation with anti-CD8 antibody (Fig. 7 and data not shown).

View larger version (67K): [in this window] [in a new window]   FIG. 7.   Increase of tyrosine phosphorylation of cellular signaling molecules upon stimulation with an anti-CD8 antibody. A total of 5 × 106 cells were incubated without () or with (+) an anti-CD8 antibody at 37°C for 1 min and lysed with lysis buffer. Precleared cell lysates were used for immunoprecipitation (I.P.) with an antibody as indicated at the top. Immunoprecipitates were immunoblotted (I.B.) with an antiphosphotyrosine antibody (P-Y). After that, each immunoblot was stripped and reprobed with the specific antibody against cellular protein to show the equivalent level of protein expression (bottom of each panel). Lanes: 1 and 2, CD8; 3 and 4, CD8-K1-C; 5 and 6, CD8-D1; 7 and 8, CD8-D2; 9 and 10, CD8-YY/FF; 11 and 12, CD8-Y282F; 13 and 14, CD8-TYF. Arrows indicate the individual cellular proteins and CD8-K1 chimeras; asterisks indicate the immunoglobulin heavy chain of anti-CD8 antibody used for stimulation. Sizes are indicated in kilodaltons.

Phosphorylation of tyrosine residues in the ITAM sequence of K1. In addition to increased tyrosine phosphorylation of cellular signaling molecules upon stimulation, it was evident that immune complexes of Syk, p85, and Vav contained an additional tyrosine-phosphorylated 37-kDa protein from stimulated cells expressing CD8-K1-C, or CD8-Y₂₈₂F (Fig. 7, lanes 4 and 12). CD8-K1-C and CD8-Y₂₈₂F chimeras have the same molecular mass of 37 kDa. To investigate tyrosine phosphorylation of CD8-K1 chimeras upon stimulation, cell lysates were used for immunoprecipitation with anti-CD8 antibody, which was followed by immunoblotting with antiphosphotyrosine antibody. Tyrosine phosphorylation was detected from the CD8-K1-C chimera upon stimulation, while it was not detected from CD8 under the same conditions (Fig. 8A). Additionally, tyrosine phosphorylation of mutant CD8-K1 chimeras was not detected upon antibody stimulation with the exception of CD8-Y₂₈₂F, which was weakly tyrosine phosphorylated after stimulation (Fig. 8A, lane 16). These results indicate that tyrosine residues within the ITAM sequence of K1 were phosphorylated upon stimulation, as is seen with ITAM sequences of cellular immune receptors (3, 8). These observations also suggest that the tyrosine residues of CD8-K1-C and CD8-Y₂₈₂F become phosphorylated upon stimulation and subsequently bind to SH2-containing cellular effectors, including Syk, p85, and Vav, as shown in Fig. 7.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   Tyrosine phosphorylation of CD8-K1 chimeras upon stimulation and by cellular tyrosine kinases. (A) Tyrosine phosphorylation of CD8-K1 chimeras upon stimulation. A total of 5 × 106 BJAB cells expressing CD8, CD8-K1-C, or mutant forms of CD8-K1 were incubated with (+) or without () and anti-CD8 antibody at 37°C for 1 min and lysed with lysis buffer. Precleared cell lysates were used for immunoprecipitation (I.P.) with an anti-CD8 antibody. Immunoprecipitates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (I.B.) with an antiphosphotyrosine antibody (P-Y). Lanes: 1 and 2, CD8; 3 and 4, CD8-K1-C; 5 and 6, CD8-D1; 7 and 8, CD8-D2; 9 and 10, CD8-D3; 11 and 12, CD8-YY/FF; 13 and 14, CD8-TYF; 15 and 16, CD8-Y282F. (B) Tyrosine phosphorylation of the ITAM of K1 by cellular tyrosine kinases. COS-1 cells were cotransfected with pcDNA3-CD8-K1-C together with a tyrosine kinase expression vector; 48 h after transfection, COS-1 cells were lysed with lysis buffer. Precleared cell lysates were used for immunoprecipitation with an anti-CD8 antibody. Immunoprecipitates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and reacted with an antiphosphotyrosine antibody. Lanes: 1, no DNA; 2, CD8-K1-C; 3, Syk; 4, CD8-K1-C with Src; 5, CD8-K1-C with Lck; 6, CD8-K1-C with Fyn; 7, CD8-K1-C with Lyn; 8, CD8-K1-C with Syk; 9, CD8-K1-C with Zap70. Expression of CD8-K1-C is shown at the bottom. (C) Tyrosine phosphorylation of CD8-K1 chimeras by Syk in COS-1 cells. COS-1 cells were cotransfected with a Syk expression vector together with an expression vector containing CD8-K1 chimeras; 48 h after transfection, lysates were immunoprecipitated with an anti-CD8 antibody and then immunoblotted with an antiphosphotyrosine antibody. Lanes: 1, no DNA; 2, CD8-K1-C; 3, Syk; 4, CD8 with Syk; 5, CD8-K1-C with Syk; 6, CD8-YY/FF with Syk; 7, CD8-Y282F with Syk; lane 8, CD8-TYF with Syk; 9, CD8-D1 with Syk; 10, CD8-D2 with Syk. Arrows indicate locations of CD8-K1 chimeras, and asterisks indicate the immunoglobulin heavy and light chains. Sizes are indicated in kilodaltons.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

KSHV contains a distinct open reading frame called K1 at a position equivalent to the gene encoding the STP of HVS. The oncogenic potential of the KSHV K1 gene has been unambiguously demonstrated in rodent fibroblasts and in primary lymphocytes (18). In this report, we present compelling evidence that the ITAM in the cytoplasmic region of K1 is capable of transducing signals that elicit cellular activation events. In addition, mutational analyses demonstrate that the ITAM of K1 is important for its signal-transducing activity.

The biochemical signaling event immediately following BCR stimulation is the induction of tyrosine phosphorylation of a number of cellular proteins (3). As for BCR stimulation, antibody cross-linking of CD8 rapidly induced tyrosine phosphorylation in cells expressing the CD8-K1 chimera. The absence of tyrosine phosphorylation in cells expressing mutant forms of CD8-K1 chimeras resulted from the loss of the conserved tyrosine residues of the ITAM. We also demonstrate that a single motif of YXX(L/P) within the ITAM of K1 is likely to be sufficient for the induction of cellular tyrosine phosphorylation, while both motifs of the ITAM are necessary for the induction of intracellular free-calcium mobilization. However, the CD8-D3 chimera which contains both YXX(L/P) motifs but has a deletion of the negatively charged conserved region is incapable of inducing cellular tyrosine phosphorylation after stimulation. This finding indicates that a residue immediately upstream of the YXX(L/P) motif is also necessary for the induction of tyrosine phosphorylation.

Systematic searches for the optimal sequences for binding to SH2 domains have shown that individual members of SH2-containing proteins select unique tyrosine-containing partners (33, 34). Comparisons of the ITAM sequence of K1 with optimal recognition sequences for the SH2 domain reveal that the first YYSL motif of K1 is similar to the recognition sequences for SH2 domains of Lyn, Fgr, Syk, and Shc, whereas the second YTQP motif is similar to those of Abl, Crk, Nck, Grb2, and Vav. This may explain at least in part the differential effect of K1 mutations on the association with different signal-transducing proteins in Fig. 7. Further studies are needed to identify additional cellular signal-transducing molecules associated with K1, which will provide a detailed understanding of the signal transduction pathway mediated by the KSHV K1.

Despite the presence of a proline residue instead of leucine in the distal YXXL motif, the ITAM of K1 shares functional properties with those of Ig and Ig. The first tyrosine residue in the ITAMs of K1 and Ig (8) is likely to be the major site for phosphorylation, and little or no tyrosine phosphorylation is observed at the second tyrosine residue in the ITAM. Additionally, both conserved tyrosine residues in the ITAMs of K1, Ig, and Ig (8) are necessary to induce intracellular calcium mobilization. As shown for Ig and Ig (3), the nonligated resting ITAM of CD8-K1 chimeras may associate with Src family tyrosine kinases. Upon stimulation with antibody, Src family kinases are activated to phosphorylate the tyrosine residues in the ITAM of the CD8-K1 chimera. Tyrosine phosphorylation of the ITAM of K1 then leads to recruitment of Syk, Vav, PI3-kinase, and perhaps some other SH2 domain-containing effector.

Aggregation of the LMP1 of EBV through its six membrane-spanning domains has been shown to mimic tumor necrosis factor receptor aggregation, generating constitutive signals that results in pleiotropic effects, including the activation of NF-B and cell growth (25). The HVS STP has also been found to be present as an oligomerized form through the collagen motif (unpublished results). In fact, a mutation which disrupts the collagen repeats has been shown to disrupt the transforming activity of STP-C488 (13). Recently, we have shown that the extracellular region of K1 forms disulfide-linked oligomers (18). However, the degree of oligomerization varied greatly with the source of the K1 gene (18). In the case of cytokine receptors, oligomerization occurs as a results of ligand binding, and it is the ligand-mediated oligomerization per se that is responsible for the recruitment of signaling molecules to recognition sequences in the cytoplasmic region of the oligomerized receptor (11). It remains to be determined whether signaling through sequence in the cytoplasmic domain of K1 (Fig. 9) is constitutively active, as it appears to be with LMP1 of EBV and STP of HVS, or whether signaling is induced by some unidentified ligand. The long extracellular domain of K1 (Fig. 9), the extensive sequence variation in the extracellular domains of K1s from different sources, and the variation in the degree of oligomerization of K1s from different sources (18) are likely to be important clues for subsequent studies aimed at obtaining a closer picture of the role of K1 in different disease states.

View larger version (15K): [in this window] [in a new window]   FIG. 9.   Schematic representation of signal transduction of the KSHV K1. The topology of two K1 molecules is shown to represent oligomerization. The amino-terminal extracellular region shows the possible disulfide linkage, and Y-P indicates the phosphorylated tyrosine residue in ITAM. The transmembrane domain of K1 is depicted by a vertical cylinder. PLC, phospholipase c-.