Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells.
Mammalian procholecystokinin (pro-CCK) is known to have three sulfated tyrosine residues, one of which is present in the CCK 8 moiety and two additional residues present in the carboxyl-terminal extension. In the present study, inhibition of tyrosine sulfation by sodium chlorate decreased the secretion of processed CCK 8 in CCK-expressing endocrine cells in culture. It was then demonstrated that when each of these tyrosines individually, as well as all three together, was mutated to phenylalanine and expressed in endocrine cells, CCK was still processed and secreted. However, the amount of CCK secreted varied with the type of mutation. Substitution of Phe to Tyr in CCK 8 reduced the quantity of secreted CCK 8 by 50%, and when all the sulfated Tyr were mutated to Phe the quantity of secreted CCK was reduced by about 70%, similar to what is observed with chlorate treatment. Changing of the putative phosphorylation site serine to alanine does not affect the processing. Serine phosphorylation at this site may play a functional role in regulatory events. Our results demonstrate that tyrosine sulfation alters the amount of secretion but is not an absolute requirement for the processing and secretion of CCK in this cell line. Tyrosine sulfation of CCK may still be important for its solubility, stabilization, and/or functional interaction.
Vishnuvardhan
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Department of Pharmacology & Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Beinfeld
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MC
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002704:Chlorates / Q000494:pharmacology; D002766:Cholecystokinin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D002850:Chromatography, Gel; D003470:Culture Media / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D002704:Chlorates; D003470:Culture Media; D011498:Protein Precursors; D013431:Sulfates; D014443:Tyrosine; D012694:Serine; C060249:procholecystokinin; D002766:Cholecystokinin; C032706:sodium chlorate