Characterization of the mouse bile salt export pump overexpressed in the baculovirus system

Citation
J. Noe et al., Characterization of the mouse bile salt export pump overexpressed in the baculovirus system, HEPATOLOGY, 33(5), 2001, pp. 1223-1231
Citations number
54
Categorie Soggetti
Gastroenerology and Hepatology","da verificare
Journal title
HEPATOLOGY
ISSN journal
02709139 → ACNP
Volume
33
Issue
5
Year of publication
2001
Pages
1223 - 1231
Database
ISI
SICI code
0270-9139(200105)33:5<1223:COTMBS>2.0.ZU;2-4
Abstract
The bile salt export pump (Bsep), a member of the ATP-binding cassette supe rfamily of transporters, mediates the ATP-dependent canalicular secretion o f bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein i n Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation s ites for protein kinase A, protein kinase C (PKC) and Ca2+-calmodulin depen dent kinase II, we have also tested whether mBsep undergoes phosphorylation . MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than con trol membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinit y and is the most potent inducer of ATPase activity, Cyclosporin A, glibenc lamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt-stimulated ATPase activity. MBsep exists as a phosph o-protein when expressed in Sf9 cells and the immunoprecipitated mBsep comp lex is a substrate for the catalytic subunit of PKC. When mBsep and the alp ha -isoform of mouse PKC are co-expressed in Sf9 cells, a ninefold stimulat ion of phosphorylation occurs. This is further increased to 18-fold after a ctivation by phorbol ester. Given that bile salts activate selected PKC iso forms in hepatocytes, including the alpha isoform, the phosphorylation of m Bsep by PKC alpha may represent a point of regulation for this transporter that is mediated by its own substrate.