Interactome of transforming growth factor-β type I receptor (TβRI):: Inhibition of TGFβ signaling by Epac1
Interactome of transforming growth factor-β type I receptor (TβRI):: Inhibition of TGFβ signaling by Epac1
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DOI：
10.1021/pr060427q
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发表时间：
2007-01-05
影响因子：
4.4
通讯作者：
Souchelnytskyi, Serhiy
中科院分区：
文献类型：
--
作者：
Conrotto, Paolo;Yakymovych, Ihor;Souchelnytskyi, Serhiy
转化生长因子-β（TGF β）是细胞生长、分化和凋亡的有效调节剂。I型TGF β受体（T β RI）是TGF β启动细胞内信号传导的关键受体。在这里，我们报告的蛋白质组学为基础的识别与T β RI形成复合物的蛋白质。使用2D-GE和MALDI-TOF质谱，我们确定了16个蛋白质特异性相互作用的GST融合的T β RI Thr204Asp结构与组成型活性丝氨酸/苏氨酸激酶。我们使用免疫印迹证实了cAMP调节的鸟嘌呤核苷酸交换因子1（Epac 1），α-血影蛋白，PIASy和α-连环蛋白的受体的相互作用。受体与Epac 1的相互作用需要T β RI的完整激酶活性，但不受Epac 1 cAMP结合结构域缺失的影响。TGF β 1诱导的Smad2的C-末端磷酸化在体内和体外Epac 1的存在下被抑制。Epac1还抑制TGF β 1/T β RI依赖的转录激活，如荧光素酶报告基因测定所评价。我们观察到Epac 1的表达抵消了TGF β/T β RI依赖的细胞粘附减少和TGF β/T β RI诱导的细胞迁移刺激。因此，我们已经报道了新的T β RI相互作用蛋白，并表明Epac1抑制TGF β依赖的细胞迁移和粘附调节。
Transforming growth factor-beta (TGF beta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGF beta receptor (T beta RI) is the key receptor for initiation of intracellular signaling by TGF beta. Here we report proteomics-based identification of proteins that form a complex with T beta RI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused T beta RI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), alpha-spectrin, PIASy, and alpha-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of T beta RI but was not affected by deletion of cAMP-binding domain of Epac1. TGF beta 1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGF beta 1/T beta RI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGF beta/T beta RI-dependent decrease of cell adhesion and TGF beta/T beta RI-induced stimulation of cell migration. Thus, we have reported novel T beta RI-interacting proteins and have shown that Epac1 inhibited TGF beta-dependent regulation of cell migration and adhesion.