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. 2003 Jul;77(14):7924-35.
doi: 10.1128/jvi.77.14.7924-7935.2003.

Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation

Shan-Lu Liu  1 , Michael I Lerman, A Dusty Miller
Affiliations

Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation

Shan-Lu Liu et al. J Virol. 2003 Jul.

Abstract

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.

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Figures

FIG. 1.
FIG. 1.
JSRV Env constructs and their transforming activities. Shown at the top is the JSRV Env protein which is processed into the SU and TM subunits of the mature Env as indicated. The presumed endoplasmic reticulum signal peptide (SP), membrane-spanning domain (M), and cytoplasmic tail (CT) are shown. In FLAG-Jenv, the first 72 amino acid residues of JSRV Env were replaced by the signal sequence of preprotrypsin followed by a FLAG sequence (SS-FLAG). A similar construct was also created for ENTV Env, FLAG-Eenv (not shown). Jenv-FLAG is identical to the wild-type Env, except that a FLAG sequence was attached to its cytoplasmic tail. JSRV-MLV and JSRV-HIV-1 are two chimeras in which the cytoplasmic tails of JSRV Env were replaced with those of 10A1-MLV and HIV-1, respectively. JSU-hIgG was designed to express JSRV SU-human IgG Fc fusion protein as described previously (19). Shown at right are the transforming activities of each construct measured using the 2-week transformation assay.
FIG. 2.
FIG. 2.
Effects of mutations in the cytoplasmic tails of JSRV and ENTV Env proteins on 208F cell transformation. (A) Sequence alignment of the Env cytoplasmic tails of JSRV7 (AF357971), JSRV21 (AF105220), JSRVSA (M80216), ENTV (Y16627), and ESRV (AF153615). Amino acid numbering is based on that of JSRVSA. YXXM motifs present in all JSRV and ENTV strains are boxed, tyrosine residues are in bold, and E584 and MKY 588-590, which are absent in ESRV, are underlined. (B and C) Transforming activities of JSRV and ENTV Env mutants. Transformed foci were counted 2 weeks after transfection. Jenv-FLAG and FLAG-Eenv were used as positive controls, while 10A1-MLV Env served as a negative control. Results are means ± standard deviations of three independent experiments. (D) Flow cytometric analysis of ENTV Env protein expression. 208F cells harvested immediately after the 2-week transformation assay in G418 were incubated with mouse anti-FLAG antibody on ice for 2.5 h, washed, and incubated with FITC-conjugated mouse secondary antibody on ice for 45 min, followed by FACS analysis.
FIG. 3.
FIG. 3.
PI3K-dependent Akt activation in 208F cells transformed by JSRV or ENTV Env proteins. (A) In vitro Akt kinase assay. Cells were incubated in serum-free DMEM overnight. The cells were then incubated for 4 h in DMEM containing 0, 25, or 50 μM LY or 50 μM LY plus 5% FBS (lane labeled “50*”). Akt activity was measured by incubating the immunoprecipitated Akt with substrate GSK-3 in the presence of 200 μM ATP, followed by immunoblotting with antibody against phospho-GSK-3α/β. Total Akt resulting from the same immunoprecipitations were determined by anti-Akt antibody. (B) Detection of Akt phosphorylation. Lysates of serum-starved cells were subjected to SDS-PAGE followed by immunoblotting with anti-phospho-Akt (Ser473) to detect Akt phosphorylation or using anti-Akt to determine the total amount of Akt present.
FIG. 4.
FIG. 4.
The PI3K specific inhibitor LY reverses the transformed phenotypes of cells expressing JSRV and ENTV Env proteins. 208F cells transformed by JSRV Env, ENTV Env, or Fos were treated with 25 μM LY dissolved in DMSO or with an equal amount of DMSO (final concentration, 0.25%) only for 4 days, with media containing the drug changed every day (see text for details). Untreated 208F cells are shown at left.
FIG. 5.
FIG. 5.
The Env proteins of JSRV and ENTV are not tyrosine phosphorylated in 208F cells transformed by these proteins. (Left panel) 208F cells transformed by JSRV (Jenv-FLAG) or ENTV (FLAG-Eenv) Env proteins were serum-starved overnight, cell lysates were harvested and were immunoprecipitated (IP) by using anti-FLAG antibody, and immunoprecipitates were immunoblotted (IB) by using antiphosphotyrosine antibody 4G10. Lysate from EGF-stimulated A431 cells (A431/EGF) (Upstate Biotechnology) was run as a positive control. (Right panel) The same PVDF membrane was then stripped and reblotted by using anti-FLAG antibody to ensure that the IP worked properly.
FIG. 6.
FIG. 6.
JSRV and ENTV Env proteins do not interact with PI3K or Grb2. (A and B) GST pull-down assay. Cell lysates harvested from the JSRV (A) or ENTV (B) Env-transformed 208F cells were incubated with GST-Grb2 (full length), GST-p85, or GST alone at 4°C for 4 h, and the resulting precipitates were subjected to SDS-PAGE followed by immunoblotting by using 4G10 or anti-FLAG antibody. Before cell lysis, JSRV Env-transformed cells were treated with phosphatase inhibitors (2 mM sodium orthovanadate and 2 mM hydrogen peroxide) to enhance the detection of phosphorylated proteins, while ENTV Env-transformed cells were not so treated. Lanes labeled “Lysate” contain cell lysates that were not subjected to GST IP. (C) Cell lysate from JSRV Env-transformed 208F cells (not treated with phosphatase inhibitors) was subjected to SDS-PAGE before (lane 1) or after (lane 2) IP by using anti-FLAG antibody and were immunoblotted by using anti-p85. Lysate from EGF-stimulated A431 cells (Upstate Biotechnology) was run as a control (lane 3). (D) Lysate from JSRV Env-transformed 208F cells (not treated with phosphatase inhibitors) was analyzed before (lane 1) or after (lane 2) immunoprecipitation using anti-p85 by immunoblotting by using anti-FLAG.
FIG. 7.
FIG. 7.
Akt is activated in 208F cells transformed by JSRV or ENTV Env tyrosine mutants, as determined by Akt phosphorylation at Ser473 (pAkt) and in vitro kinase assay (pGSK-3α/β). Experimental procedures were the same as those described in Fig. 3, except that the cell lysates were obtained from 208F cells transformed by several tyrosine mutants of JSRV and ENTV Env. 208F cells transformed by the parental JSRV (Jenv-FLAG) or ENTV (FLAG-Eenv) Env proteins served as positive controls. The lower panel shows Env expression in the cell lysates by immunoblotting with the anti-FLAG antibody. Similar results were also obtained for all other ENTV and JSRV Env tyrosine mutants, and all experiments were performed at least three times.
FIG. 8.
FIG. 8.
MAPK phosphorylation was not detected in 208F cells transformed by JSRV or ENTV Env proteins or their tyrosine mutants. Twenty micrograms of each cell lysate was used for SDS-PAGE analysis followed by immunoblotting by using anti-phospho-MAPK or anti-MAPK. pp44 and pp42 represent phospho-Thr202/Tyr204 MAPK, while p44 and p42 represent total cellular MAPK. (A) Cells were serum-starved (DMEM only) overnight. (B) Cells were grown in normal culture medium (DMEM with 5% FBS).
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