Brooks S F, Herget T, Broad S, Rozengurt E
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
J Biol Chem. 1992 Jul 15;267(20):14212-8.
We have examined the regulation of expression of 80K/MARCKS, a major and specific protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts. Addition of bombesin (10 nM) to confluent quiescent cultures of these cells induced a dramatic and sustained down-regulation of 80-kDa mRNA and protein levels to a minimum of 5% of control within 8 and 48 h, respectively, without depletion of PKC activity. In contrast, the effect of phorbol 12,13-dibutyrate on 80K/MARCKS mRNA levels was transient, and recovery of these transcripts correlated with the loss of PKC activity. The ability of bombesin to down-regulate 80K/MARCKS mRNA levels was dose-dependent (ED50 0.5 nM) and was abolished by both the specific bombesin antagonist [Leu13 psi (CH2NH),Leu14]bombesin and by prior depletion of PKC. Of a range of agents tested, platelet-derived growth factor (PDGF), but not insulin or Ca2+ ionophore, also down-regulated 80K/MARCKS mRNA to 24% of control within 5 h. Prior down-regulation of PKC abolished the effect of PDGF at a concentration of 7 ng/ml. Surprisingly, at higher doses (25 ng/ml), PDGF induced the down-regulation of 80K/MARCKS mRNA in a PKC-independent manner. Furthermore, elevation of cAMP, either through receptor-mediated mechanisms (e.g. prostaglandin E1) or by direct stimulation of adenylate cyclase (e.g. forskolin), also caused a marked dose-dependent depletion of 80K/MARCKS mRNA levels, which were further reduced by co-administration with cAMP-phosphodiesterase inhibitors. The rate of transcription of the 80K/MARCKS gene was unaltered by treatment of cells with either bombesin, PDGF, or forskolin/1-methyl-3-isobutylxanthine. These results indicate a role for both PKC-dependent and -independent pathways in growth factor-induced down-regulation of 80K/MARCKS expression, through a post-transcriptional mechanism.
我们研究了瑞士3T3成纤维细胞中主要且特异性的蛋白激酶C（PKC）底物80K/MARCKS的表达调控。向这些细胞的汇合静止培养物中添加铃蟾肽（10 nM），分别在8小时和48小时内诱导80 kDa mRNA和蛋白水平急剧且持续下调至对照水平的最低5%，而PKC活性未耗竭。相比之下，佛波醇12,13 - 二丁酸酯对80K/MARCKS mRNA水平的影响是短暂的，这些转录本的恢复与PKC活性的丧失相关。铃蟾肽下调80K/MARCKS mRNA水平的能力是剂量依赖性的（半数有效剂量为0.5 nM），并且被特异性铃蟾肽拮抗剂[Leu13 ψ(CH2NH),Leu14]铃蟾肽以及预先耗竭PKC所消除。在一系列测试的因子中，血小板衍生生长因子（PDGF），而非胰岛素或钙离子载体，也在5小时内将80K/MARCKS mRNA下调至对照水平的24%。预先下调PKC消除了7 ng/ml浓度的PDGF的作用。令人惊讶的是，在更高剂量（25 ng/ml）时，PDGF以PKC非依赖性方式诱导80K/MARCKS mRNA的下调。此外，通过受体介导机制（如前列腺素E1）或直接刺激腺苷酸环化酶（如福斯可林）升高cAMP，也导致80K/MARCKS mRNA水平显著的剂量依赖性降低，与cAMP磷酸二酯酶抑制剂共同给药时进一步降低。用铃蟾肽、PDGF或福斯可林/1 - 甲基 - 3 - 异丁基黄嘌呤处理细胞，80K/MARCKS基因的转录速率未改变。这些结果表明，通过转录后机制，PKC依赖性和非依赖性途径在生长因子诱导的80K/MARCKS表达下调中均起作用。
