Related Experiment Videos
Multiple ras downstream pathways mediate functional repression of the homeobox gene product TTF-1
C Missero1, M T Pirro, R Di Lauro
1Stazione Zoologica "A. Dohrn" Villa Comunale, 80121 Naples, Italy.
Abstract:
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.
Insights
Oncogenic Ras inactivates thyroid transcription factor-1 (TTF-1) via the Raf/MEK/ERK pathway and another Ras pathway, inhibiting thyroid cell differentiation. This study elucidates Ras signaling in thyroid cancer.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- Oncogenic Ras expression in thyroid cells suppresses thyroid-specific genes and inactivates TTF-1.
- Understanding Ras downstream pathways is crucial for thyroid cancer development.
Purpose of the Study:
- To investigate how Ras signal transduction pathways affect TTF-1 transcriptional activity.
- To identify specific Ras effector pathways involved in suppressing thyroid cell differentiation.
Main Methods:
- Transient-transfection assays using Ras effector mutants.
- Analysis of TTF-1 transcriptional activity.
- In vitro and in vivo phosphorylation studies of TTF-1 by ERK.
- Inhibition studies using MEK inhibitors (U0126, PD98059).
Main Results:
- Ras mutant V12S35, interacting with Raf, inhibits TTF-1 activity.
- Activated Raf (Raf-BXB) inhibits TTF-1; MEK inhibitors partially rescue TTF-1 activity, implicating the ERK pathway.
- ERK directly phosphorylates TTF-1 at three serine residues, abolishing its activity.
- A distinct Ras pathway involving V12N38 Ras also represses TTF-1 activity independently of ERK.
- Combined activation of Raf and V12N38 Ras leads to near-complete TTF-1 inactivation.
Conclusions:
- The Raf/MEK/ERK cascade partially mediates Ras-induced TTF-1 inactivation.
- An additional, uncharacterized Ras pathway contributes to TTF-1 repression.
- Coordinated action of these pathways inhibits TTF-1 function and thyroid cell differentiation.