Cejudo-Martin Pilar, Yuen Angela, Vlahovich Nicole, Lock Peter, Courtneidge Sara A, Díaz Begoña
Cancer Center, Tumor Microenvironment and Metastasis Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
La Trobe Institute for Molecular Science, La Trobe University, Victoria, Australia.
PLoS One. 2014 Sep 26;9(9):e107674. doi: 10.1371/journal.pone.0107674. eCollection 2014.
Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5'RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5β. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5β has a short unique amino terminal sequence encoded by the newly discovered exon 6β; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5β mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5β is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene.
Tks5是一种支架蛋白和Src底物，通过其在侵袭小体形成和功能中的重要作用参与细胞迁移和基质降解。我们之前曾描述过，Tks5对于斑马鱼神经嵴细胞在体内的迁移至关重要。在本研究中，我们试图通过分析编码Tks5的基因sh3pxd2a的小鼠突变体来研究Tks5在哺乳动物发育中的功能。通过在小鼠中进行基因捕获对sh3pxd2a基因进行纯合破坏导致新生死亡，并出现继发腭完全裂开。有趣的是，来自纯合基因捕获sh3pxd2a小鼠的胚胎成纤维细胞仅缺少在蛋白质提取物中观察到的特征性Tks5三联体的最高分子量条带，而较低分子量条带未受影响。这一发现，连同存在两个缺少SH3PXD2A前5个外显子的人类表达序列标签，使我们推测在第V内含子中存在第二个替代转录起始位点。我们对小鼠成纤维细胞进行了5'RACE，并分离出sh3pxd2a基因的一个新转录本，其编码一种新型Tks5异构体，我们将其命名为Tks5β。这种新型异构体与Tks5的长形式不同，它缺少赋予对磷脂酰肌醇-3,4-二磷酸亲和力的PX结构域。相反，Tks5β具有由新发现的外显子6β编码的短独特氨基末端序列；该外显子包括一个起始密码子，位于外显子6的5'端29 bp处。Tks5β mRNA在MEF和所有分析的小鼠成年组织中表达。Tks5β是Src酪氨酸激酶的底物，其表达通过蛋白酶体降解途径进行调节。总之，这些发现表明较大的Tks5异构体对于正确的哺乳动物发育至关重要，以及sh3pxd2a基因的转录复杂性。
