Localization of the viral and cellular Src kinases to perinuclear vesicles in fibroblasts

Previous studies have shown that the viral oncogene product, v-Src, is localized to a perinuclear structure. Here, we demonstrate by immunofluorescence analysis that the perinuclear structure corresponds to a local concentration of vesicles. Overexpressed normal cellular Src, c-Src, and a temperature-sensitive mutant of v-Src also are associated with these perinuclear vesicles. This perinuclear localization is observed in a variety of cell types using several different antibodies to Src, and it is independent of the fixation method. Immunoelectron ultracryomicroscopic analysis of rat fibroblast cells transformed by v-Src demonstrates an association of this protein with the limiting membranes of vesicles concentrated in the perinuclear region. These vesicles appear at the electron microscopic level to be multivesicular bodies on the basis of their size (0.3-1.0 microns in diameter), large electron-transparent lumens, and electron-dense vesicular inclusions. Morphometric analysis indicates that approximately 20% of the total cell v-Src protein is associated with these structures. This subpopulation of v-Src may have been recovered from the plasma membrane via the endocytotic pathway in a manner analogous to endocytosis of the epidermal growth factor receptor. Localization of the Src tyrosine kinases to these perinuclear endocytotic vesicles may be necessary for oncogenic transformation by v-Src and for normal functions of c-Src.