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Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus - PubMed

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Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus

H Lee et al. Mol Cell Biol. 1998 Sep.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

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Figures

FIG. 1
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 (−), CD8Δ (Δ), 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.

FIG. 2
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.

FIG. 3
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) CD8Δ (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).

FIG. 4
FIG. 4

Surface expression of IgM and induction of intracellular free-calcium concentration. (A) Level of IgM surface expression. BJAB expressing CD8Δ 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.

FIG. 5
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) CD8Δ; (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.

FIG. 6
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, CD8Δ; 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.

FIG. 7
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 (αP-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, CD8Δ; 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.

FIG. 8
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 CD8Δ, 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 (αP-Y). Lanes: 1 and 2, CD8Δ; 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, CD8Δ 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.

FIG. 9
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. PLCγ, phospholipase c-γ.

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