TIMAP is a positive regulator of pulmonary endothelial barrier function.
TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.
Csortos
Csilla
C
Department of Medical Chemistry, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
Czikora
Istvan
I
Bogatcheva
Natalia V
NV
Adyshev
Djanybek M
DM
Poirier
Christophe
C
Olah
Gabor
G
Verin
Alexander D
AD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D008840:Microfilament Proteins / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D017931:DNA Primers; D007527:Isoenzymes; D008565:Membrane Proteins; D008840:Microfilament Proteins; C459597:PPP1R16B protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C071058:moesin; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; C517102:PPP1CC protein, human; D054645:Protein Phosphatase 1; D013917:Thrombin