Pyne N J, Pyne S
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, Scotland.
Cell Signal. 1997 May-Jun;9(3-4):311-7. doi: 10.1016/s0898-6568(96)00190-8.
We have investigated the mechanisms regulating p38MAPK in airway smooth muscle cells. Incubation of cells with platelet-derived growth factor (PDGF) stimulated MAPKA kinase-2 activity, a kinase immediately down-stream of P38MAPK. Preincubation of the cells with forskolin (10 microM), which stimulated a 20-fold increase in intracellular cAMP formation, inhibited this response. An antibody raised against subdomain VI of yeast Ste20 kinase co-immunoprecipitated p38MAPK from cell lysates. The immunoprecipitated kinase(s) was shown to catalyse the phosphorylation of myelin basic protein (MBP) and to activate purified MAPKAP kinase-2. Incubation of cells with PDGF did not increase the amount of p38MAPK isolated in the anti-Ste20 immunoprecipitate. However, the kinase phosphorylated MBP and stimulated purified MAPKAP kinase-2 activity more effectively than kinase from control cells. The preincubation of cells with forskolin (10 microM) reduced the amount of P38MAPK in the immunoprecipitate and this correlated with a decrease in kinase activity. We conclude the PDGF induces the activation of p38MAPK, whereas forskolin elicits its dissociation from the complex with Ste20. Therefore, Ste20/p38MAPK may form part of a signal transduction pathway linked to activation of MAPKAP kinase-2 in ASM cells.
我们研究了气道平滑肌细胞中调节p38丝裂原活化蛋白激酶（p38MAPK）的机制。用血小板衍生生长因子（PDGF）孵育细胞可刺激MAPK激酶-2的活性，该激酶是P38MAPK下游紧邻的激酶。用福斯可林（10微摩尔）预孵育细胞，可刺激细胞内cAMP生成增加20倍，从而抑制了这一反应。一种针对酵母Ste20激酶亚结构域VI产生的抗体可从细胞裂解物中共同免疫沉淀p38MAPK。经证明，免疫沉淀的激酶可催化髓鞘碱性蛋白（MBP）的磷酸化，并激活纯化的MAPK激活的蛋白激酶-2（MAPKAP激酶-2）。用PDGF孵育细胞不会增加抗Ste20免疫沉淀中分离出的p38MAPK的量。然而，该激酶比对照细胞中的激酶更有效地磷酸化MBP并刺激纯化的MAPKAP激酶-2的活性。用福斯可林（10微摩尔）预孵育细胞可减少免疫沉淀中P38MAPK的量，这与激酶活性的降低相关。我们得出结论，PDGF诱导p38MAPK的激活，而福斯可林引发其与Ste20复合物的解离。因此，Ste20/p38MAPK可能构成与气道平滑肌细胞中MAPKAP激酶-2激活相关的信号转导途径的一部分。
