Multiple Ras downstream pathways mediate functional repression of the homeobox gene product TTF-1
Multiple Ras downstream pathways mediate functional repression of the homeobox gene product TTF-1
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DOI：
10.1128/mcb.20.8.2783-2793.2000
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发表时间：
2000-04-01
影响因子：
5.3
通讯作者：
Di Lauro, R
中科院分区：
文献类型：
--
作者：
Missero, C;Pirro, MT;Di Lauro, R
致癌Ras在甲状腺细胞中的表达导致几种甲状腺特异性基因表达的丧失和TTF-1的失活，TTF-1是甲状腺正常发育所需的含有同源结构域的转录因子。为了了解Ras下游的信号转导途径如何参与抑制分化表型，我们测试了Ras效应区突变体在瞬时转染试验中影响TTF-1转录活性的能力。我们发现，V12S35 Ras，一种已知与Raf特异性相互作用但不与RaIGDS或磷脂酰肌醇3-激酶（PI3激酶）相互作用的突变体，抑制TTF-1活性。Raf的活化形式（Raf-BXB）的表达也以类似程度抑制TTF-1功能，而MEK抑制剂U0126和PD98059部分缓解Ras介导的TTF-1失活，表明细胞外信号调节激酶（ERK）途径参与该过程。事实上，ERK直接磷酸化TTF-1在三个丝氨酸残基，并伴随这些丝氨酸突变为丙氨酸完全废除ERK介导的磷酸化在体外和体内。由于Raf/MEW/ERK通路的激活仅占致癌Ras对TTF-1引起的活性的一部分，因此其他下游通路可能参与该过程。我们发现PI3激酶、Rho、Rac和RaIGDS的激活对TTF-1的转录活性没有影响。然而，一个不好表征的pas突变体，V12N38 Ras，可以通过ERK非依赖性途径部分抑制TTF-1的转录活性。重要的是，组成型激活的Raf和V12N38 Ras的伴随表达导致TTF-1活性几乎完全丧失。我们的数据表明Raf/MEK/ERK级联可能与V12N38 Ras激活的尚未表征的信号通路协同作用，以抑制TTF-1功能并最终抑制甲状腺细胞分化。
Expression of oncogenic Ras in thyroid cells results in loss of expression of several thyroid-specific genes and inactivation of TTF-1, a homeodomain-containing transcription factor required for normal development of the thyroid gland. In an effort to understand how signal transduction pathways downstream of Ras may be involved in suppression of the differentiated phenotype, we have tested mutants of the Ras effector region for their ability to affect TTF-1 transcriptional activity in a transient-transfection assay. We find that V12S35 Ras, a mutant known to interact specifically with Raf but not with RaIGDS or phosphatidylinositol 3-kinase (PI3 kinase) inhibits TTF-1 activity. Expression of an activated form of Raf (Raf-BXB) also inhibits TTF-1 function to a similar extent, while the MEK inhibitors U0126 and PD98059 partially relieve Ras-mediated inactivation of TTF-1, suggesting that the extracellular signal-regulated kinase (ERK) pathway is involved in this process. Indeed, ERK directly phosphorylates TTF-1 at three serine residues, and concomitant mutation of these serines to alanines completely abolishes ERK-mediated phosphorylation both in vitro and in vivo. Since activation of the Raf/MEW/ERK pathway accounts for only part of the activity elicited by oncogenic Ras on TTF-1, other downstream pathways are likely to be involved in this process. We find that activation of PI3 kinase, Rho, Rac, and RaIGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized pas mutant, V12N38 Ras, can partially repress TTF-1 transcriptional activity through an ERK-independent pathway. Importantly, concomitant expression of constitutive activated Raf and V12N38 Ras results in almost complete loss of TTF-1 activity, Our data indicate that the Raf/MEK/ERK cascade may act in concert with an as-yet-uncharacterized signaling pathway activated by V12N38 Ras to repress TTF-1 function and ultimately to inhibit thyroid cell differentiation.