ZETA-PKC INDUCES PHOSPHORYLATION AND INACTIVATION OF I-KAPPA-B-ALPHA IN-VITRO
ZETA-PKC INDUCES PHOSPHORYLATION AND INACTIVATION OF I-KAPPA-B-ALPHA IN-VITRO
复制标题
DOI：
10.1002/j.1460-2075.1994.tb06578.x
复制
发表时间：
1994-06-15
期刊：
影响因子：
11.4
通讯作者：
MOSCAT, J
中科院分区：
文献类型：
--
作者：
DIAZMECO, MT;DOMINGUEZ, I;MOSCAT, J
蛋白激酶C（xi 3-PKC）的xi同种型是一种不能结合佛波醇酯的独特PKC，在非洲爪蟾卵母细胞和哺乳动物细胞中NF-xi B活化以及促有丝分裂信号传导过程中是必需的。为了研究xi PKC控制细胞功能的机制，将该酶在大肠杆菌中表达为与麦芽糖结合蛋白（MBP）的融合蛋白，以允许固定在直链淀粉珠上，从而研究细胞提取物中可能与xi PKC形成复合物的信号蛋白。获得了与NF-κ B/I κ B途径相互作用的以下证据。MBP-xi PKC，而不是MBP，结合并激活一种潜在的新的I κ B激酶，其分子量约为50 kDa，能够调节I κ B-α功能。I κ B激酶的激活依赖于PKC酶活性和ATP，表明xi PKC直接或间接控制功能显著的I κ B激酶的活性。重要的是，来自TNF-α刺激的NM-3 T3成纤维细胞的xi PKC免疫沉淀物显示出比未处理的对照组更高的I κ B磷酸化活性，表明这些发现的体内相关性。我们还表明，在这里，xi PKC协会和激活MKK-MAPK在体外，这表明其中一个机制，即过度表达xi PKC导致失调的细胞生长可能占至少部分由激活的MKK-MAPK复合物。然而，无论是MKK还是MAPK都不负责推定的I κ B磷酸化活性。这些数据为理解xi PKC的功能迈出了决定性的一步。
The xi isotype of protein kinase C (xi 3-PKC), a distinct PKC unable to bind phorbol esters, is required during NF-xi B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by xi PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es), with xi PKC. The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained. MBP-xi PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of -50 kDa molecular weight able to regulate I kappa B-alpha function. Activation of the I kappa B kinase was dependent on PKC enzymatic activity and ATP, suggesting that xi PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase. Importantly, xi PKC immunoprecipitates from TNF-alpha-stimulated NM-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings. We also show here that xi PKC associates with and activates MKK-MAPK in vitro, suggesting that one of the mechanisms whereby overexpression of xi PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex. However, neither MKK nor MAPK is responsible for the putative I kappa B phosphorylating activity. These data provide a decisive step towards understanding the functions of xi PKC.