Phosphorylation of beta-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells.
Phosphorylation of beta-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells.
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DOI：
10.1152/ajpcell.00096.2008
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发表时间：
2008-05
期刊：
影响因子：
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通讯作者：
S. Taurin;N. Sandbo;Douglas M. Yau;N. Sethakorn;N. Dulin
中科院分区：
文献类型：
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作者：
S. Taurin;N. Sandbo;Douglas M. Yau;N. Sethakorn;N. Dulin
细胞外 ATP 通过激活 G 蛋白偶联 P2Y 嘌呤受体来刺激血管平滑肌细胞 (VSMC) 增殖。我们之前已经证明 ATP 刺激蛋白激酶 A (PKA) 的瞬时激活，与已建立的嘌呤能受体有丝分裂信号一起促进 VSMC 增殖 (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004)。我们还表明，PKA 可以在体外和过表达细胞模型（Taurin S、Sandbo N、Qin Y、Browning D、Dulin NO. J Biol Chem 281：9971-9976，2006）中在两个新位点（Ser552 和 Ser675）磷酸化 β-连环蛋白。 beta-Catenin 通过激活 T 细胞因子 (TCF) 转录因子家族来促进细胞增殖，这些转录因子驱动与细胞周期进程相关的基因转录，包括细胞周期蛋白 D1。在本研究中，使用针对 β-连环蛋白磷酸化 Ser552 或磷酸化 Ser675 位点的磷酸特异性抗体，我们发现 ATP 可以刺激这两个位点内源性 β-连环蛋白的 PKA 依赖性磷酸化，而不影响其在 VSMC 中的表达水平。这转化为通过增加磷酸化（通过 PKA）β-连环蛋白与 TCF-4 的关联来对 TCF 转录活性进行 PKA 依赖性刺激。使用 PKA 抑制剂 PKI 或显性失活 TCF-4 突变体，我们发现 ATP 诱导的细胞周期蛋白 D1 启动子激活、细胞周期蛋白 D1 蛋白表达和 VSMC 增殖均依赖于 PKA 和 TCF 活性。总之，我们展示了一种通过 PKA 磷酸化内源性 β-连环蛋白进行调节的新模式，并证明了这种机制对于 ATP 诱导的 VSMC 增殖的重要性。
Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.