Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma
Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma
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DOI：
10.1002/ijc.25893
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发表时间：
2011-11-01
影响因子：
6.4
通讯作者：
Brummer, Tilman
中科院分区：
文献类型：
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作者：
Eisenhardt, Anja E.;Olbrich, Heike;Brummer, Tilman
毛细胞星形细胞瘤（PA）是一种Ras/Raf/MEK/ERK信号失调的肿瘤实体。在PA中观察到的常见遗传病变与ERK通路活性异常有关，包括NF1失活、KRAS或BRAF功能获得突变。为了更详细地研究编码Ser/ thr激酶B-Raf的原癌基因的突变谱，我们分析了来自PA儿童的64个原发肿瘤样本，其中包括2例1型神经纤维瘤病（NF1）患者。已知的BRAF（V600E）突变在6/64（9.38%）的样本中被发现。我们首次报道了在NF1患者中同时存在体细胞BRAFV（600E）突变，这表明在PA中不止一种Ras/ERK通路成分可能受到影响。此外，2/64（3.13%）的样本在BRAF中携带一个3 bp的插入，导致苏氨酸599重复。这个保守的残基位于激活片段内，如果以ras依赖的方式磷酸化，则在Raf激活中起关键作用。在这里，我们证明了这个突变体（B-Raf(insT)）和另一个B-Raf突变体，在这个位置携带两个额外的苏氨酸残基，显示出与B-Raf（V600E）相当的体外激酶活性和细胞MEK/ERK激活电位。值得注意的是，缬氨酸残基取代苏氨酸对B-Raf活性有类似的影响，这表明肽骨架被额外的氨基酸扭曲，而不是额外的潜在磷酸化位点的插入，破坏了激酶结构域的非活性构象。我们还证明B-Raf（insT）和B-Raf(V600E)，而不是B-Raf(wt)，会引起人类星形胶质细胞的剧烈形态学改变。
Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain-of-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAFV(600E) mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf(insT)) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E). Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf(insT) and B-Raf(V600E), but not B-Raf(wt), provoke drastic morphological alterations in human astrocytes.