Identification of the 52 kDa cytoskeletal-like protein of cytochalasin D-stimulated normal rat kidney (NRK/CD) cells as substrate-associated glycoprotein p52 [plasminogen-activator inhibitor type-1 (PAI-1)]. Expression of p52 (PAI-1) in NRK/CD cells is regulated at the level of mRNA abundance.
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.
Higgins
P J
PJ
Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, NY 12208.
Ryan
M P
MP
D000818:Animals; D015153:Blotting, Western; D015638:Cytochalasin D / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D007668:Kidney / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats
D015849:Plasminogen Inactivators; D012333:RNA, Messenger; D015638:Cytochalasin D; D004247:DNA