Röhrkasten A, Meyer H E, Nastainczyk W, Sieber M, Hofmann F
Institut für Physiologische Chemie, Medizinische Fakultät, Universität des Saarlandes, Homburg/Saar, Germany.
J Biol Chem. 1988 Oct 25;263(30):15325-9.
The cAMP-dependent phosphorylation of the 165-kDa subunit of the receptor for organic calcium channel blockers (CaCB-receptors) was studied. Tryptic peptide analysis showed that cAMP-dependent protein kinase phosphorylates rapidly a serine in one peptide. Up to three peptides containing phosphoserine and -threonine are phosphorylated in a 2-h incubation. The isolated 165-kDa subunit was digested with trypsin and the endoproteinase Lys-C and Glu-C. The rapidly phosphorylated peptide was isolated from each digest. The amino acid sequence was determined by Edman degradation and compared with the deduced amino acid sequence of the CaCB-receptor from rabbit skeletal muscle (Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 238, 313-318). Phosphoserine was determined as the phenylthiohydantoin-derivative of dithiothreitol-dehydroalanine. The phosphorylated serine was identified as Ser-687 which is localized between the transmembrane regions II and III. A second phosphopeptide was isolated into which phosphate was incorporated into Ser-1617 with a slow time course. This peptide is located in the COOH-terminal cytoplasmic domain of the 165-kDa subunit. It is anticipated that phosphorylation of serine 687 affects the opening probability of the calcium channel.
对有机钙通道阻滞剂受体（CaCB受体）165 kDa亚基的环磷酸腺苷（cAMP）依赖性磷酸化进行了研究。胰蛋白酶肽分析表明，cAMP依赖性蛋白激酶可迅速使一个肽段中的丝氨酸磷酸化。在2小时的孵育过程中，多达三个含有磷酸丝氨酸和磷酸苏氨酸的肽段会被磷酸化。将分离出的165 kDa亚基用胰蛋白酶、内肽酶Lys-C和Glu-C进行消化。从每种消化产物中分离出快速磷酸化的肽段。通过埃德曼降解法测定氨基酸序列，并与兔骨骼肌CaCB受体的推导氨基酸序列进行比较（田边，T.，竹岛，H.，三上，A.，弗洛克齐，V.，高桥，H.，神川，K.，小岛，M.，松尾，H.，广濑，T.，和沼田，S.（1987年）《自然》238，313 - 318）。磷酸丝氨酸被确定为二硫苏糖醇脱氢丙氨酸的苯硫代乙内酰脲衍生物。磷酸化的丝氨酸被鉴定为位于跨膜区II和III之间的Ser - 687。分离出了第二个磷酸肽段，磷酸以较慢的时间进程掺入到Ser - 1617中。该肽段位于165 kDa亚基的COOH末端胞质结构域。预计丝氨酸687的磷酸化会影响钙通道的开放概率。
