Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium

Alexander Dmitriyevich Verin, Peiyi Wang, Joe G N Garcia

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We have previously shown that myosin-specific phosphatase 1 (PPase 1) activity is critical for maintaining endothelial cell barrier function (Verin et al. [1995] Am. J. Physiol. 269:L99-L108). To further characterize myosin- specific PPase 1 in endothelium, we generated antibodies specific to published sequences of the myosin-associated PPase 1 regulatory subunit (M110) from smooth muscle. Peptide antigens were designed based upon consensus sequences for a single ankyrin repeat (ANK 110) and a leucine zipper motif region (LZ 110), which represents putative sites for binding the PPase 1 catalytic subunit (CS1) and myosin, respectively. Our initial study demonstrated that each antibody immunoprecipitated 2 proteins with an apparent Mr of 110 and 70 kD on SDS-PAGE. The CS1δ isoform, which appeared to be characteristic for the myosin-specific phosphatase, was co- immunoprecipitated under non-denaturing conditions with ANK110 and LZ110 as was actin, myosin, and myosin light chain kinase (MLCK). Similarly, immunoprecipitation with specific anti-myosin or anti-MLCK antibodies under the same conditions, followed by immunostaining with either LZ110 or ANK110 revealed the same 110 and 70 kD protein bands. The 70 kD protein (p70) was immunoreactive with ANK 110 and LZ 110, was complexed with myosin and MLCK, and was detected in non-denaturing M110 immunoprecipitates. Consistent with these results, endothelial cell fractionation demonstrates the presence of p70 in both cytoskeletal and myosin-enriched fractions, but not in the myosin-depleted (cytosolic) fractions. These data suggest that endothelial cells may exhibit two distinct myosin-specific PPase 1 regulatory subunits which share certain structural features with the M110 regulatory subunit from smooth muscle and which are tightly associated with myosin and MLCK in a functional complex.

Original languageEnglish (US)
Pages (from-to)489-498
Number of pages10
JournalJournal of Cellular Biochemistry
Volume76
Issue number3
DOIs
StatePublished - Feb 14 2000
Externally publishedYes

Fingerprint

Myosin-Light-Chain Phosphatase
Myosins
Phosphoric Monoester Hydrolases
Endothelium
Myosin-Light-Chain Kinase
Endothelial Cells
Endothelial cells
Smooth Muscle
Antibodies
Ankyrin Repeat
Cell Fractionation
Leucine Zippers
Proteins
Consensus Sequence
Muscle
Immunoprecipitation
Actins
Polyacrylamide Gel Electrophoresis
Catalytic Domain
Protein Isoforms

Keywords

  • Endothelium
  • Myosin
  • Phosphatase 1 regulatory subunits

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium. / Verin, Alexander Dmitriyevich; Wang, Peiyi; Garcia, Joe G N.

In: Journal of Cellular Biochemistry, Vol. 76, No. 3, 14.02.2000, p. 489-498.

Research output: Contribution to journalArticle

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