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P52(PAI-1) and actin expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells.

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.

SUBMITTER: Higgins PJ

PROVIDER: S-EPMC1151528 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Project description:Cell shape profoundly affects cellular metabolic activity, protein and nucleic acid synthesis, and cytoskeletal organization. To examine the influence of cell shape on protein expression, normal rat kidney (NRK) cells were exposed to the microfilament-disrupting drug cytochalasin D (CD), labelled with [35S]methionine, and newly synthesized cellular and cytoskeletal proteins examined by two-dimensional gel electrophoresis. CD produced dramatic changes in cell shape (from a flat to round phenotype) with concomitant 3-7-fold increases in the cellular content and cytoskeletal deposition of the microfilament-associated proteins actin, alpha-actinin, and tropomyosin isoform 1. Augmented actin protein content in NRK/CD cells was paralleled by a corresponding increase in actin mRNA abundance and was inhibited by prior addition of actinomycin D. A detergent-insoluble protein of 52 kDa was also detected at high levels in the cytoskeletal fraction of NRK/CD cells. Two-dimensional electrophoretic mapping of total cellular and cytoskeletal proteins revealed this 52 kDa protein to be the previously described glycoprotein p52 [Higgins & Ryan (1989) Biochem. J. 257, 173-182]. By using electrophoretic and immunochemical criteria, p52 was identified as plasminogen-activator inhibitor type-1 (PAI-1). Like actin, CD-induced p52(PAI-1) synthesis, cellular content, and partitioning to the detergent-insoluble cytoskeletal compartment reflected a corresponding increase in p52(PAI-1) mRNA. Such induction was similarly inhibited by actinomycin D. p52(PAI-1) expression in the NRK-cell system is thus responsive to CD-mediated shape changes and requires ongoing RNA synthesis for its induction. Differential extraction of detached cell bodies and the substrate-adherent 'remnant' fraction of NRK/CD cultures, furthermore, indicated that p52(PAI-1) was not an intrinsic internal cytoskeletal element but, rather, selectively localized to the extracellular residue. p52(PAI-1) retained its detergent-insoluble characteristics even in this isolated 'remnant' fraction, where it was also the predominant protein species resolved.

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P52(PAI-1) and actin expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells.

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