Premium
Characterization of the protein phosphatase 1 catalytic subunit in endothelium: Involvement in contractile responses
Author(s) -
Verin Alexander D.,
Csortos Csilla,
Durbin Steve D.,
Aydanyan Antonina,
Wang Peiyi,
Patterson Carolyn E.,
Garcia Joe G.N.
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/1097-4644(2000)79:1<113::aid-jcb110>3.0.co;2-9
Subject(s) - protein subunit , phosphatase , protein phosphatase 2 , protein phosphatase 1 , microbiology and biotechnology , chemistry , biochemistry , biology , phosphorylation , gene
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 (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, overexpression 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. J. Cell. Biochem. 79:113–125, 2000. © 2000 Wiley‐Liss, Inc.