Mutational analysis of the cytoplasmic tail of jaagsiekte sheep retrovirus envelope protein.
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.
Hull
Stacey
S
Cancer Research Institute, Sprague Hall, University of California Irvine, Irvine, CA 92697-3900, USA.
Fan
Hung
H
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002472:Cell Transformation, Viral / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry / Q000821:virology; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D015686:Gene Products, env / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D022141:Jaagsiekte sheep retrovirus / Q000502:physiology*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D015686:Gene Products, env; D011494:Protein Kinases; C546843:mTOR protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1