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    J Cell Biochem. 2000 Jul 19;79(1):113-25.

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

    Verin AD, Csortos C, Durbin SD, Aydanyan A, Wang P, Patterson CE, Garcia JG.

    Source

    Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. averin@welch.jhu.edu

    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, I-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 (alpha, delta, and gamma) in human and bovine EC. To characterize the myosin-associated EC CS1 isoform, myosin-enriched bovine EC fraction was screened with anti-CS1alpha and anti-CS1delta antibodies The anti-CS1delta antiserum, but not anti-CS1alpha antiserum cross reacts with the CS1 isoform present in myosin-enriched fraction and CS1delta was found in stable association with EC myosin/myosin light chain kinase (MLCK) complex in MLCK immunoprecipitates under nondenaturing conditions. Consistent with these data, overexpression of CS1delta-GFP construct in bovine endothelium followed by immunoprecipitation of CS1 with anti-GFP antibody revealed the stable association of CS1delta with actomyosin complex. Finally, screening of a human EC oligo(dT)-primed cDNA library with a probe encoding a rat CS1delta cDNA segment yielding several positive clones that encoded the entire CS1delta open reading frame and partially noncoding regions. Sequence analysis determined a high homology ( approximately 99%) with human CS1delta derived from a teratocarcinoma cell line. Together, these data suggest that CS1delta is the major of PPase 1 isoform specifically associated with EC actomyosin complex and which participates in EC barrier regulation.

    Copyright 2000 Wiley-Liss, Inc.

    PMID:
    10906760
    [PubMed - indexed for MEDLINE]

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