Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.
Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.
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DOI：
10.1007/s11010-013-1823-3
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发表时间：
2014-01
影响因子：
4.3
通讯作者：
Boden SD
中科院分区：
文献类型：
--
作者：
Sangadala S;Yoshioka K;Enyo Y;Liu Y;Titus L;Boden SD
骨骼系统和其他器官的发育和修复高度依赖于骨形态发生蛋白（BMP）通路的精确调节。临床上使用BMP诱导骨形成的部分限制是灵长类动物需要比细胞培养物或啮齿类动物更高剂量的重组蛋白。因此，增加细胞对BMP的反应性已成为我们关注的焦点。我们确定成骨LIM矿化蛋白LMP-1与Smurf 1（Smad泛素调节因子1）相互作用，并阻止Smads的泛素化，从而增强BMP活性。在负责骨形成的LMP-1区域中，存在直接与Smurf 1 WW 2结构域相互作用的基序，从而有效地竞争与BMP途径中的关键信号蛋白Smad 1和Smad 5的结合。在这里，我们发现，同一区域还包含一个基序，与Jun激活结构域结合蛋白1（Jab 1）相互作用，靶向一个共同的Smad，Smad 4，由BMP和转化生长因子-β（TGF-β）途径共享，用于蛋白酶体降解。Jab 1首先被鉴定为转录因子c-Jun的共激活因子，它与TGF-β超家族的关键细胞内信号分子Smad 4、Smad 5和Smad 7结合，并引起这些Smads的泛素化和/或降解。我们证实了Jab 1与LMP-1的直接相互作用，使用重组表达的野生型和突变体蛋白质在狭缝印迹结合试验。我们假设LMP-1与Jab 1的结合阻止了这些Smads的结合和随后的降解，从而导致成骨Smads在细胞中的积累增加。我们确定了LMP-1中的序列基序，该基序基于已知与Jab-1相互作用的几种细胞信号分子的MAME/MAST序列分析预测与Jab 1相互作用。我们进一步突变LMP-1中潜在的关键相互作用残基，并在体外结合试验中显示与Jab 1结合的丧失。在小鼠成肌细胞中使用BMP-反应性荧光素酶报告基因和碱性磷酸酶测定法评价了各种野生型和突变型LMP-1蛋白的活性，所述成肌细胞分化为成骨细胞表型。最后，为了加强LMP-1和Jab 1相互作用的生理相关性，我们表明，在BMP处理后，LMP-1的过表达引起Smad 4的核积累，这反映了细胞中Smad信号的增加。
Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquiti-nation and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding assays in vitro. The activities of various wild-type and mutant LMP-1 proteins were evaluated using a BMP-responsive luciferase reporter and alkaline phosphatase assay in mouse myoblastic cells that were differentiated toward the osteoblastic phenotype. Finally, to strengthen physiological relevance of LMP-1 and Jab1 interaction, we showed that overexpression of LMP-1 caused nuclear accumulation of Smad4 upon BMP treatment which is reflective of increased Smad signaling in cells.