Sippel C J, Fallon R J, Perlmutter D H
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 Jul 29;269(30):19539-45.
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.
一种肝细胞胆小管磷蛋白（大鼠肝脏胆小管胆汁酸转运体/胞外ATP酶/细胞黏附分子105）的cDNA转染可赋予异源细胞胆汁酸外排和胞外ATP酶活性（西佩尔，C.J.，苏奇，F.J.，阿南塔拉亚南，M.，和珀尔马特，D.H.（1993年）《生物化学杂志》268卷，2083 - 2091页）。我们之前的研究还表明，该转运体的磷酸化程度与其胆汁酸外排活性之间存在正相关。在本研究中，我们在该转运体胞质尾内的蛋白激酶C依赖性（T502A、S503A）和酪氨酸激酶依赖性（Y488F）磷酸化共有序列中引入氨基酸残基的位点特异性突变，以确定体内磷酸化的位点并研究每个位点的功能意义。使用pCDM8表达载体将突变体和野生型构建体转染到COS细胞中。代谢标记和细胞表面标记表明，突变蛋白的合成和转运到细胞表面的效率与野生型相同。使用转染细胞裂解物进行的磷酸氨基酸分析表明，T502A、S503A突变体含有[32P]磷酸酪氨酸，Y488F突变体含有[32P]磷酸丝氨酸，野生型含有两种32P标记的氨基酸，证明在对照条件下Ser503和Tyr488是该系统中仅有的磷酸化氨基酸。用T502A、S503A突变体cDNA转染的细胞中胆汁酸转运活性完全丧失，而用Y488F突变体cDNA转染的细胞中胆汁酸转运活性得以保留，但动力学特性发生了改变，尽管这两种构建体赋予胞外ATP酶活性的程度与野生型cDNA相同。综上所述，这些数据表明该转运体的胆汁酸外排活性需要Ser503的位点特异性磷酸化，并受Tyr488的位点特异性磷酸化调控。
