P21 Ras heterogeneous localization in transformed cells

Abstract

The cellular targets for the Kirsten murine sarcoma virus (KiMSV)-transforming protein, p21ras, are unknown. Other studies have indicated that the mature form of p21 is distributed diffusely on the cytoplasmic face of the plasma membrane. However, after fixation without buffer washes, indirect immunofluorescent staining of sparse cultures revealed a particularly well preserved cellular architecture and a strikingly heterogeneous subcellular distribution of p21 in transformed normal rat kidney (NRK) cells but not in their untransformed counterparts. The transformed cells included A KiMSV-transformed NRK line. NRK cells newly transformed with KiMSV. A temperature-sensitive (ts) KiMSV-transformed NRK line. An uninfected, spontaneously transformed NRK line in which p21 was neither phosphorylated nor overproduced. In the tsKNRK line p21 was abundant at both permissive and non-permissive temperatures; however, its distribution was heterogeneous at the permissive temperature only. Observation of this array of cells indicates that the transformation-associated p21 distribution does not require overexpression of the gene, nor phosphorylation of the protein, nor the viral oncogene. Furthermore, it is reversible in the tsKNRK cells, and so appears to be highly correlated with acquisition of a transformed morphology. Accumulations of p21 occurred preferentially in subcellular locations similar to those where ruffles were observed by phase contrast microscopy and lamellar and villous extensions were observed by scanning electron microscopy (SEM). Since enhanced ruffling is a morphological correlate of transformation in a variety of cells, the distribution of p21 observed here may relate to its function as a transforming molecule.