Abstract
Flat revertants of v-ras-transformed (KNRK) rat kidney cells, which express elevated levels of p21ras protein, were generated to high efficiencies with sodium butyrate (NaB). Overall protein synthesis in revertants was not different from parental cells, although changes were evident in expression and distribution of specific microfilament-associated cytoskeletal proteins. Quantitative two-dimensional electrophoresis revealed revertant-associated 3-4-fold increases in cytoskeletal deposition of the microfilament-associated proteins gelsolin and vinculin correlating with microfilament reorganization and focal-contact formation respectively. Similar increases in actin content were evident at both the total-cellular- and cytoskeletal-associated-protein levels. In contrast, intermediate-filament family elements (vimentin, lamins) remained unaltered. The only unique protein resolved in flat revertants was p52, a 52 kDa extracellular-matrix-associated protein previously identified as plasminogen-activator inhibitor type 1 (PAI-1). p52(PAI-1) expression was induced early during generation of the revertant phenotype and preceded development of focal-contact structures. NaB-induced p52(PAI-1) synthesis and generation of early morphological reversion in KNRK cells required ongoing RNA synthesis, since exposure to actinomycin D before addition of NaB inhibited both events. p52(PAI-1) induction by NaB was regulated at the level of mRNA abundance; in contrast, actin mRNA levels were the same in parental and revertant cells, suggesting that the increased actin content which typified the revertant phenotype was due to augmented actin microfilament stability.