Vishnuvardhan D, Beinfeld M C
Department of Pharmacology & Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Biochemistry. 2000 Nov 14;39(45):13825-30. doi: 10.1021/bi0011072.
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.
已知哺乳动物前胆囊收缩素（pro-CCK）有三个硫酸化酪氨酸残基，其中一个存在于CCK 8部分，另外两个残基存在于羧基末端延伸区。在本研究中，氯酸钠抑制酪氨酸硫酸化降低了培养的CCK表达内分泌细胞中加工后的CCK 8的分泌。随后证明，当这些酪氨酸中的每一个单独以及全部三个一起突变为苯丙氨酸并在内分泌细胞中表达时，CCK仍被加工和分泌。然而，分泌的CCK量随突变类型而变化。CCK 8中苯丙氨酸被酪氨酸取代使分泌的CCK 8量减少50%，当所有硫酸化酪氨酸都突变为苯丙氨酸时，分泌的CCK量减少约70%，这与氯酸盐处理时观察到的情况相似。将假定的磷酸化位点丝氨酸突变为丙氨酸不影响加工过程。该位点的丝氨酸磷酸化可能在调节事件中发挥功能作用。我们的结果表明，酪氨酸硫酸化改变了分泌量，但不是该细胞系中CCK加工和分泌的绝对必要条件。CCK的酪氨酸硫酸化可能对其溶解性、稳定性和/或功能相互作用仍然很重要。
