title: Molecular characterization of myosin phosphatase in endothelium.
abstract: The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1β) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1β or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1β suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1β. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton.
authors.affiliation: Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA.
authors.forename: Kyung-Mi
authors.initials: KM
authors.lastname: Kim
authors.forename: Csilla
authors.initials: C
authors.lastname: Csortos
authors.forename: Istvan
authors.initials: I
authors.lastname: Czikora
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: Nagavedi S
authors.initials: NS
authors.lastname: Umapathy
authors.forename: Gabor
authors.initials: G
authors.lastname: Olah
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002199:Capillary Permeability; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C086680:ETV5 protein, human; D007527:Isoenzymes; D008565:Membrane Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014157:Transcription Factors; C071776:radixin; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human