Missero C, Pirro M T, Di Lauro R
Stazione Zoologica "A. Dohrn" Villa Comunale, 80121 Naples, Italy.
Mol Cell Biol. 2000 Apr;20(8):2783-93. doi: 10.1128/MCB.20.8.2783-2793.2000.
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 RalGDS 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/MEK/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 RalGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized Ras 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.
致癌性Ras在甲状腺细胞中的表达导致几个甲状腺特异性基因的表达缺失以及TTF-1的失活，TTF-1是一种含同源结构域的转录因子，对甲状腺的正常发育至关重要。为了了解Ras下游的信号转导通路如何参与分化表型的抑制，我们在瞬时转染实验中测试了Ras效应区域的突变体影响TTF-1转录活性的能力。我们发现V12S35 Ras，一种已知特异性与Raf相互作用而不与RalGDS或磷脂酰肌醇3激酶（PI3激酶）相互作用的突变体，可抑制TTF-1活性。活化形式的Raf（Raf-BXB）的表达也能在相似程度上抑制TTF-1功能，而MEK抑制剂U0126和PD98059可部分缓解Ras介导的TTF-1失活，这表明细胞外信号调节激酶（ERK）通路参与了这一过程。事实上，ERK直接在三个丝氨酸残基处磷酸化TTF-1，并且将这些丝氨酸同时突变为丙氨酸在体外和体内均完全消除了ERK介导的磷酸化。由于Raf/MEK/ERK通路的激活仅占致癌性Ras对TTF-1引发活性的一部分，其他下游通路可能也参与了这一过程。我们发现PI3激酶、Rho、Rac和RalGDS的激活对TTF-1转录活性没有影响。然而，一个特性不明的Ras突变体V12N38 Ras可通过一条不依赖ERK的通路部分抑制TTF-1转录活性。重要的是，组成型活化Raf和V12N38 Ras的共表达导致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激活的一条尚未明确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。
