Human T-cell leukemia virus type 1 Tax protein interacts with and mislocalizes the PDZ domain protein MAGI-1

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). HTLV-1 encodes the oncoprotein Tax1, which is essential for immortalization of human T-cells and persistent HTLV-1 infection in vivo. Tax1 has a PDZ binding motif (PBM) at its C-terminus. This motif is crucial for the transforming activity of Tax1 to a T-cell line and persistent HTLV-1 infection. Tax1 through the PBM interacts with PDZ domain proteins such as Dlg1 and Scribble, but it has not been determined yet, which cellular PDZ proteins mediate the functions of Tax1 PBM. Here we demonstrate that Tax1 interacts with the PDZ domain protein MAGI-1 in a PBM-dependent manner, and the interaction mislocalizes MAGI-1 from the detergent-soluble to the detergent-insoluble cellular fraction in 293T cells and in HTLV-1-infected T-cells. In addition, Tax1-transformation of a T-cell line from interleukin (IL)-2-dependent to IL-2-independent growth selects cells with irreversibly reduced expression of MAGI-1 at mRNA level. These findings imply that Tax1, like other viral oncoproteins, targets MAGI-1 as a mechanism to suppress its anti-tumor functions in HTLV-1-infected cells to contribute to the transforming activity of T-cells and persistent HTLV-1 infection.

Figure 1

Tax1 interacts with MAGI‐1 in a PBM‐dependent manner. (A) Structures of Tax1, Tax1ΔC and Tax2 proteins used in this study. The amino acid sequence of the PBM is shown. (B, C) 293T cells were transiently transfected with pHβPr‐neo‐Tax1 (lane 2), pHβPr‐neo‐Tax1ΔC (lane 3) or pHβPr‐neo plasmid (lane 1) together with (C) or without pcDNA3.1:FLAG‐MAGI‐1c (B) by the lipofection method (FUGENE 6). At 48 h post‐transfection, the cells were treated with lysis buffer, and cell lysates immunoprecipitated with anti‐Tax1 antibody. Total cell lysates (Input) and immunoprecipitates (IP: Tax1) were characterized by a Western blotting analysis using the indicated antibodies.

Figure 2

Subcellular localization of Tax1 and endogenous MAGI‐1 in 293T cells. 293T cells were transfected with either the Tax1 or Tax1ΔC plasmid as described in the methods. The cells were stained with anti‐Tax1 (red), anti‐MAGI‐1 (green), and Hoechst 33258 (blue) for nuclear staining. The stained cells were examined by fluorescent light microscopy.

Figure 3

Tax1 translocates MAGI‐1 from the soluble to the insoluble cellular fraction. (A) The 293T cells transfected with Tax1 (lane 2), Tax1ΔC (lane 3) or control (lane 1) were divided into two groups. One group was treated with the NP40 lysis buffer and fractionated as described in Materials and Methods. The other group was directly treated with the sodium dodecyl sulfate (SDS)‐sample buffer and used as the total fraction. The three types of samples were size‐fractionated by SDS‐polyacrylamide gel electrophoresis (PAGE) followed by a Western blotting analysis. (B) Cell lysates from the total, soluble and insoluble fractions of Jurkat and SLB‐1 cells were prepared as describe in (A) and the amounts of MAGI‐1, Tax1, Tubulin and Lamin B proteins in the lysates were measured by a Western blotting analysis.

Figure 4

MAGI‐1 downregulation by interleukin (IL)‐2‐independent transformation of T‐cells. (A) CTLL‐2 and Jurkat cells were transfected with lentiviruses encoding Tax1. At 48 h after transfection, cell lysates were prepared and the amounts of MAGI‐1, Tax1 and Tubulin were determined by a Western blotting analysis. (B) CTLL‐2 cells stably expressing Tax1 established as previously described15 were used as IL‐2‐dependent cells (lane 2–9). All the above cells were then transferred to IL‐2 deficient medium and cultured for more than 1 month. Only one Tax1 expressing clone (Tax1–18) survived in the absence of IL‐2 (right panel, lane 10). Cell lysates were prepared from the indicated cells, and protein expression was measured by a Western blotting analysis. (C) CTLL‐2 cells transformed by Tax1 and Tax1ΔC were established as described previously.34 Cell lysates were prepared from these Tax1‐transformed (lanes 2–6), Tax1ΔC‐transformed (lanes 7–9) and parental CTLL‐2 cells (lane 1), and the expressions of MAGI‐1, Tax1, Syntrophin‐β　 and Tubulin proteins were measured by a Western blotting analysis. (D) A set of clones each transformed by either Tax1 or Tax1ΔC was selected from (C) above, and the relative gene expression of MAGI‐1 was evaluated by the quantitative real‐time polymerase chain reaction (PCR) method. The amounts of MAGI‐1 were normalized to those of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression. The quantitative results are expressed as mean ± standard deviation (SD) of three values per sample. The experiment was independently carried out twice to confirm reproducibility.

Figure 5

Restoration of interleukin (IL)‐2 cannot rescue MAGI‐1 expression in transformed cells. (A) Two distinct Tax1‐transformed IL‐2‐independent CTLL‐2 cells (clone 1, 3) were cultured with or without IL‐2 for 1 week, and the expression of MAGI‐1, Tax1 and Tubulin in the transformed cells with IL‐2 (lane 3, 5), the transformed cells without IL‐2 (lane 2, 4) and parental CTLL‐2 cells with IL‐2 (lane 1) were measured by a Western blotting analysis. (B) hAkt1 mΔPH‐transformed IL‐2‐independent CTLL‐2 cells were cultured with or without IL‐2 for 1 week, and the expressions of MAGI‐1, Akt1 and Tubulin proteins in the hAkt1 mΔPH‐expressing cells with IL‐2 (lane 2), the hAkt1 mΔPH‐transformed cells without IL‐2 (lane 3), the hAkt1 mΔPH‐transformed cells with IL‐2 (lane 4) as well as the vector‐transduced CTLL‐2 cells cultured in the presence of IL‐2 (lane 1) were measured by a Western blotting analysis.

Figure 6

Expression of MAGI‐1 in human T‐cell lines transformed by HTLV‐1. (A) Cell lysates were prepared from five HTLV‐1‐transformed (lanes 4–8) and three human T‐cell leukemia virus type 1 (HTLV‐1)‐negative (lanes 1–3) T‐cell lines. The lysates were subjected to Western blotting analysis with the antibodies indicated. (B) Expression of MAGI‐1 mRNA in two HTLV‐1‐negative and two HTLV‐1‐positive cell lines was measured by the quantitative real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) method. The amounts of MAGI‐1 were normalized to those of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression. The quantitative results are expressed as mean ± standard deviation (SD) of three values per sample. (C) Cell lysates were prepared from two Tax1 and two Tax2‐immortalized T‐cells (left panel) and HTLV‐1 uninfected Jurkat cells and Tax untreated peripheral blood mononuclear cells (PBMCs) (right panel). Western blotting analysis was performed using the corresponding antibodies.