Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.
Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.
复制标题
DOI：
10.1016/s0021-9258(18)41884-4
复制
发表时间：
1992-08
期刊：
影响因子：
--
通讯作者：
Victor Adler;Alla Polotskaya;Fred Wagner;Andrew S. Kraft
中科院分区：
文献类型：
--
作者：
Victor Adler;Alla Polotskaya;Fred Wagner;Andrew S. Kraft
在U937白血病细胞中加入佛波酯可刺激丝氨酸63和73上c-jun的磷酸化。为了分离刺激这种磷酸化的蛋白激酶，我们使用了肝素-琼脂糖层析，然后用谷胱甘肽-琼脂糖珠与谷胱甘肽S转移酶和c-jun的5-89位氨基酸(Gst-c-jun)的融合蛋白结合的亲和层析。通过这个过程，我们纯化了一个67 kDa的蛋白，该蛋白能够磷酸化GST-c-jun以及完整的c-jun蛋白。通过对丝氨酸63和73进行突变，然后创建与GST(GST-c-Jun mut)的融合蛋白，我们证明了这种蛋白激酶特异性地磷酸化c-Jun氨基末端的这些位置。磷酸酶2A处理纯化的c-jun氨基末端蛋白激酶(cJAT-PK)可抑制其磷酸化GST-c-jun的能力。这种失活的酶可以通过蛋白激酶C(PKC)的磷酸化而重新激活，尽管PKC不能磷酸化GST-c-Jun底物。由于v-jun在体内不能被磷酸化，我们比较了cJAT-PK与GST-v-jun和GST-c-jun mut的结合能力。CJAT-PK与GST-c-jut的结合能力是GST-v-jun的50倍，这表明v-jun中缺失的Delta结构域在cJAT-PK的结合中发挥了作用。这些结果表明，蛋白磷酸酶和PKC介导的蛋白激酶级联反应调节c-jun的磷酸化。
The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.