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.