ERK1 AND ERK2, 2 MICROTUBULE-ASSOCIATED PROTEIN-2 KINASES, MEDIATE THE PHOSPHORYLATION OF TYROSINE-HYDROXYLASE AT SERINE-31 INSITU
ERK1 AND ERK2, 2 MICROTUBULE-ASSOCIATED PROTEIN-2 KINASES, MEDIATE THE PHOSPHORYLATION OF TYROSINE-HYDROXYLASE AT SERINE-31 INSITU
复制标题
DOI：
10.1073/pnas.89.6.2365
复制
发表时间：
1992-03-15
影响因子：
11.1
通讯作者：
KREBS, EG
中科院分区：
文献类型：
--
作者：
HAYCOCK, JW;AHN, NG;KREBS, EG
酪氨酸羟化酶（TH）在体内和原位都有4个位点被磷酸化，其中3个位点（Ser 8，Ser 19，Ser 40）被磷酸化的蛋白激酶已被鉴定。在完整的细胞中，第四个位点（Ser 31）的磷酸化在佛波醇酯或神经生长因子（NGF）的作用下增加。在这里，我们表明，Ser 31被磷酸化的ERK 1和ERK 2，两个髓鞘碱性蛋白和微管相关蛋白激酶。NGF或缓激肽处理的PC 12大鼠嗜铬细胞瘤细胞的提取物在Mono Q柱上分级。TH中Ser 31的蛋白激酶活性存在于两个峰中，对应于先前鉴定为ERK 1和ERK 2的髓鞘碱性蛋白激酶活性。纯化的TH在体外的磷酸化由这两种激酶是选择性的Ser 31高达至少0.6摩尔的磷酸每摩尔TH亚基。用缓激肽或NGF处理完整的PC 12细胞增加了原位TH-Ser 31的磷酸化和ERK的催化活性（随后在体外用髓鞘碱性蛋白作为底物测量）。用染料木黄酮（一种蛋白质酪氨酸激酶抑制剂）预处理细胞可以减少缓激肽诱导的TH-Ser 31磷酸化和ERK活性的变化，但不能减少NGF诱导的TH-Ser 31磷酸化和ERK活性的变化。染料木黄酮还抑制了由佛波醇二丁酸酯、毒蕈碱和Ba 2+产生的Ser 31磷酸化的增加。这些数据表明，ERK活性是负责磷酸化TH在Ser 31在完整的细胞，并表明TH-Ser 31磷酸化可能是由多种信号通路，收敛于或之前的ERK的激活。
Tyrosine hydroxylase (TH) is phosphorylated at four sites in situ and in vivo, and the protein kinases that phosphorylate three of these sites (Ser8, Ser19, Ser40) have been identified. In intact cells, the phosphorylation of the fourth site (Ser31) is increased in response to phorbol esters or nerve growth factor (NGF). Here, we show that Ser31 is phosphorylated by ERK1 and ERK2, two myelin basic protein and microtubule-associated protein kinases. Extracts of NGF- or bradykinin-treated PC12 rat pheochromocytoma cells were fractionated on Mono Q columns. Protein kinase activity toward Ser31 in TH was present in two peaks corresponding to myelin basic protein kinase activities previously identified as ERK1 and ERK2. Phosphorylation of purified TH in vitro by both kinases was selective for Ser31 up to at least 0.6 mol of phosphate per mol of TH subunit. Treatment of intact PC12 cells with bradykinin or NGF increased both the phosphorylation of TH-Ser31 in situ and the catalytic activity of ERKs (measured subsequently in vitro with myelin basic protein as substrate). Pretreatment of the cells with genistein (a protein-tyrosine kinase inhibitor) decreased the bradykinin- but not the NGF-induced changes in both TH-Ser31 phosphorylation and ERK activity. Genistein also inhibited the increases in Ser31 phosphorylation produced by phorbol dibutyrate, muscarine, and Ba2+. The data indicate that ERK activity is responsible for phosphorylating TH at Ser31 in intact cells and suggest that TH-Ser31 phosphorylation may be regulated by multiple signaling pathways that converge at or prior to the activation of the ERKs.