Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation

C. Jeffrey Sippel, Robert Fallon, David H. Perlmutter

Research output: Contribution to journalArticle

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Abstract

Transfection of cDNA for a hepatocyte canalicular phosphoprotein, the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105, confers bile acid efflux and ecto-ATPase activities on heterologous cells (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter D. H. (1993) J. Biol. Chem. 268, 2083-2091). Our previous studies have also indicated that there is a positive correlation between the degree of phosphorylation of this transporter and its bile acid efflux activity. In this study, we introduced site-specific mutations of amino acid residues within a protein kinase C- dependent (T502A, S503A) and a tyrosine kinase-dependent (Y488F) phosphorylation consensus sequence in the cytoplasmic tail of this transporter in order to map the sites that are phosphorylated in vivo and to examine the functional significance of each. COS cells were transfected with mutant and wild type constructs using the pCDM8 expression vector. Metabolic labeling and cell surface labeling showed that the mutant proteins were synthesized and delivered to the cell surface as efficiently as the wild type. Phosphoamino acid analysis using lysates of transfected cells showed that the T502A, S503A mutant contained [32P]phosphotyrosine, the Y488F mutant contained [32P]phosphoserine, and the wild type contained both 32P-labeled amino acids, proving that Ser503 and Tyr488 are the only amino acids phosphorylated in this system under control conditions. Bile acid transport activity was completely abrogated in cells transfected with the T502A, S503A mutant cDNA and was retained but altered in kinetic characteristics in cells transfected with the Y488F mutant cDNA, even though both of these constructs conferred ecto-ATPase activity to the same extent as the wild type cDNA. Taken together, these data show that the bile acid efflux activity of this transporter requires site-specific phosphorylation of Ser503 and is regulated by site-specific phosphorylation of Tyr488.

Original languageEnglish (US)
Pages (from-to)19539-19545
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number30
StatePublished - Jul 29 1994
Externally publishedYes

Fingerprint

Phosphorylation
Bile Acids and Salts
Liver
Serine
Tyrosine
Rats
Complementary DNA
Amino Acids
Labeling
Proteins
Phosphoamino Acids
Phosphoserine
Phosphotyrosine
Phosphoproteins
Mutant Proteins
Protein-Tyrosine Kinases
Protein Kinase C
COS Cells
Consensus Sequence
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation. / Sippel, C. Jeffrey; Fallon, Robert; Perlmutter, David H.

In: Journal of Biological Chemistry, Vol. 269, No. 30, 29.07.1994, p. 19539-19545.

Research output: Contribution to journalArticle

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abstract = "Transfection of cDNA for a hepatocyte canalicular phosphoprotein, the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105, confers bile acid efflux and ecto-ATPase activities on heterologous cells (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter D. H. (1993) J. Biol. Chem. 268, 2083-2091). Our previous studies have also indicated that there is a positive correlation between the degree of phosphorylation of this transporter and its bile acid efflux activity. In this study, we introduced site-specific mutations of amino acid residues within a protein kinase C- dependent (T502A, S503A) and a tyrosine kinase-dependent (Y488F) phosphorylation consensus sequence in the cytoplasmic tail of this transporter in order to map the sites that are phosphorylated in vivo and to examine the functional significance of each. COS cells were transfected with mutant and wild type constructs using the pCDM8 expression vector. Metabolic labeling and cell surface labeling showed that the mutant proteins were synthesized and delivered to the cell surface as efficiently as the wild type. Phosphoamino acid analysis using lysates of transfected cells showed that the T502A, S503A mutant contained [32P]phosphotyrosine, the Y488F mutant contained [32P]phosphoserine, and the wild type contained both 32P-labeled amino acids, proving that Ser503 and Tyr488 are the only amino acids phosphorylated in this system under control conditions. Bile acid transport activity was completely abrogated in cells transfected with the T502A, S503A mutant cDNA and was retained but altered in kinetic characteristics in cells transfected with the Y488F mutant cDNA, even though both of these constructs conferred ecto-ATPase activity to the same extent as the wild type cDNA. Taken together, these data show that the bile acid efflux activity of this transporter requires site-specific phosphorylation of Ser503 and is regulated by site-specific phosphorylation of Tyr488.",
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