POLYAMINES ARE ESSENTIAL FOR CELL-TRANSFORMATION BY PP60(V-SRC) - DELINEATION OF MOLECULAR EVENTS RELEVANT FOR THE TRANSFORMED PHENOTYPE

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesi s, becomes upregulated during cell proliferation and transformation. H ere we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-se nsitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor al pha-difluoromethyl omithine (DFMO) to the cells (in polyamine-free med ium) before shift to permissive temperature prevented the depolymeriza tion of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro ty rosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed i n the tyrosine phosphorylation of phosphatidylinositol 3-kinase and pr oteins of 33 and 36 kD upon the temperature shift, and these hyperphos phorylations were partially inhibited by DFMO. A DFMO-sensitive increa se was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-conta ining proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to co-imm unoprecipitate with pp60v-src in a transformation-related manner. Alto gether, this model offers a tool for sorting out the protein phosphory lations and associations critical for the transformed phenotype trigge red by pp60v-src, and implicates a pivotal role for polyamines in cell transformation.