title: Characterization of the protein phosphatase 1 catalytic subunit in endothelium: involvement in contractile responses.
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.
authors.affiliation: Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. averin@welch.jhu.edu
authors.forename: A D
authors.initials: AD
authors.lastname: Verin
authors.forename: C
authors.initials: C
authors.lastname: Csortos
authors.forename: S D
authors.initials: SD
authors.lastname: Durbin
authors.forename: A
authors.initials: A
authors.lastname: Aydanyan
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: C E
authors.initials: CE
authors.lastname: Patterson
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1