Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-β signaling pathway
Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-β signaling pathway
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DOI：
10.1074/jbc.m609915200
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发表时间：
2007-06-29
影响因子：
4.8
通讯作者：
Mulder, Kathleen M.
中科院分区：
文献类型：
--
作者：
Jin, Qunyan;Ding, Wei;Mulder, Kathleen M.
我们已经确定km 23 -1作为一种新的转化生长因子β（TGF β）受体（T β R）相互作用蛋白，也是一个轻链的马达蛋白动力蛋白（动力蛋白轻链）。在此，我们通过蔗糖梯度分析证明，在TGF β存在的情况下，而不是在不存在的情况下，Km 23 -1与T β Rs一起存在于早期内体中。此外，共聚焦显微镜研究表明，内源性km 23 -1与内源性Smad 2共定位在TGF β治疗后的早期，在Smad 2易位到细胞核。此外，免疫沉淀/印迹分析表明，TGF β调节内源性km 23 -1和内源性Smad 2在体内之间的相互作用。使用小干扰RNA方法阻断Km 23 -1导致TGF β处理后总细胞内Smad 2水平和磷酸化Smad 2的细胞核水平降低。这种减少被逆转lactacystin，26 S蛋白酶体的特异性抑制剂，这表明敲低km 23 -1导致磷酸化（即激活）Smad 2的蛋白酶体降解。阻断km 23 -1还导致TGF β/Smad 2依赖性ARE-Lux转录活性降低，这被km 23 -1小干扰RNA抗性构建体所拯救。相比之下，在类似条件下，TGF β/Smad 3依赖性SBE 2-Luc转录活性没有发生降低。此外，过表达的dynactin亚基dynamitin，这是已知的破坏动力蛋白介导的细胞内运输，阻断TGF β刺激的Smad 2的核转位。总的来说，我们的研究结果首次表明，动力蛋白轻链是Smad 2依赖性TGF β信号通路所必需的。
We have identified km23-1 as a novel transforming growth factor-beta (TGF beta) receptor (T beta R)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGF beta but not in the absence, km23-1 was present in early endosomes with the T beta Rs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGF beta treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/ blot analyses showed that TGF beta regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGF beta treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGF beta/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGF beta/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGF beta-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGF beta signaling pathway.