Characterization of the protein phosphatase 1 catalytic subunit in endothelium: Involvement in contractile responses

Alexander D. Verin, Csilla Csortos, Steve D. Durbin, Antonina Aydanyan, Peiyi Wang, Carolyn E. Patterson, Joe G.N. Garcia

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

We have previously demonstrated the direct involvement of a type 1 Ser/Thr phosphatase (PPase 1) in endothelial cell (EC) barrier regulation [Am. J. Physiol. 269:L99-L108, 1995]. To further extend this observation, we microinjected either the Ser/Thr PPase inhibitor, calyculin, or the PPase 1 inhibitory protein, 1-2 into bovine pulmonary artery EC and demonstrated both an increase in F-actin stress fibers and a shift from a regular polygonal shape to a spindle shape with gaps apparent at the cell borders. Northern blot analysis with specific cDNA probes revealed the presence of three major PPase 1 catalytic subunit (CS1) isoforms (α, δ, and γ) in human and bovine EC. To characterize the myosin-associated EC CS1 isoform, myosin-enriched bovine EC fraction was screened with anti-CS1α and anti-CS1δ antibodies. The anti-CS1δ antiserum, but not anti-CS1α antiserum cross reacts with the CS1 isoform present in myosin-enriched fraction and CS1δ was found in stable association with EC myosin/myosin light chain kinase (MLCK) complex in MLCK immunoprecipitates under nondenaturing conditions. Consistent with these data, overex, pression of CS1δ-GFP construct in bovine endothelium followed by immunoprecipitation of CS1 with anti-GFP antibody revealed the stable association of CS1δ with actomyosin complex. Finally, screening of a human EC oligo(dT)-primed cDNA library with a probe encoding a rat CS1δ cDNA segment yielding several positive clOneS that encoded the entire CS1δ open reading frame and partially noncoding regions. Sequence analysis determined a high homology (≃99%) with human CS1δ derived from a teratocarcinoma cell line. Together, these data Suggest that CS1δ is the major of PPase 1 isoform specifically associated with EC actomyosin complex and which participates in EC barrier regulation. (C) 2000 Wiley-Liss, Inc.

Original languageEnglish (US)
Pages (from-to)113-125
Number of pages13
JournalJournal of Cellular Biochemistry
Volume79
Issue number1
DOIs
StatePublished - 2000

Keywords

  • CS1 isoforms in endothelium
  • Inhibitor-2
  • Microinjection

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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