Localization of the feline sarcoma virus fgr gene product (P70gag-actin-fgr): association with the plasma membrane and detergent-insoluble matrix

R Manger, S Rasheed, L Rohrschneider
1986 Journal of Virology  
The v-fgr oncogene codes for a unique transforming protein (P709ag-ac"n-fgr) that contains virus-specific determinants and cell-derived sequences for both a tyrosine-specific kinase domain and an actin domain. We examined the subcellular distribution of the v-fgr protein by immunofluorescence microscopy and various cell fractionation techniques. By immunofluorescence, the v-fgr protein was localized in a diffuse cytoplasmic pattern within transformed cells. The v-fgr protein was not detectable
more » ... was not detectable at substratum adhesion sites. Crude membrane preparations (P100) obtained fromfgr-transformed cells contained elevated levels of p70gag-achi-fgr. Further analysis of membranes on discontinous sucrose gradients revealed that p70gag-actin-fgr cofractionated with plasma membranes. Using an alternate method of fractionation, we found that the majority of the v-fgr protein remained with the insoluble matrix obtained by treating cells with a buffer containing Triton X-100. When membranes were similarly treated with detergent, nearly all of v-fgr protein remained with the residual insoluble matrix. These results suggest that the transforming activity of p70gag-actin-fgr may be directed to subcellular cytoskeletal targets at or near the cytoplasmic face of the plasma membrane. of kinase buffer (20 mM Tris hydrochloride [pH 7.5], 10 mM MnCl2); 10 ,uCi of [y-32P]ATP (3,000 Ci/mmol) was then added to this suspension. The reaction was allowed to proceed for 10 min at room temperature and was then stopped by the addition of 0.5 ml of lysis buffer containing 10 mM EDTA. Following three subsequent washes in lysis 66 on May 10, 2020 by guest
doi:10.1128/jvi.59.1.66-72.1986 fatcat:zqbvjmdwizbarab6vfeap4rggq