| Abstract |
Transforming growth factor β (TGF-β) belongs to a superfamily of cytokines
playing important role in fundamental processes of the metazoan cells such as cell
proliferation, differentiation, migration, adhesion and death. TGF-β regulates
transcriptional responses via the cytoplasmic effector proteins termed Smads. The
TGF-β signaling pathway includes the binding of TGF-β to transmembrane receptors,
the activation of Smads through phosphorylation, the homo- or hetero-oligomerization
of Smads, their translocation to the nucleus and their interaction with other nuclear
factor for the transcription of gene-targets.
The linker region of R-Smads contains many serine/threonine phosphorylation
sites which are considered to play a very important role in TGF-β signaling. The
phosphorylation of the linker by MAPKs and CDKs is the point of convergence
between the TGF-β signaling pathway and the EGF/FGF/Ras signaling pathways. The
result of this convergence as well as the general effect of the linker phosphorylation of
R-Smads on the TGF-β pathway is still under study.
The SCPs (Small CTD phosphatases) were initially identified as phosphatases of
the CTD region of RNAPII. Recent studies have associated them with the
dephosphorylation of the linker region of Smad1/2/3 and the SXS motif of Smad1.
The effect of this interaction was the attenuation of the BMP signaling but the
enhancement of TGF-β-induced transcriptional responses. The last one is opposed to
the general repression of the transcriptional machinery that had been so far observed
by SCPs.
In the present study we examined the effect of the dephosphorylation of the linker
region of Smad2/3 on the TGF-β signaling pathway. Initially, we performed a series
of titrations using increasing concentrations of expression vectors carrying the SCP1,
SCP2, SCP3 and PPM1A phosphatases in HEK293T cells. The aim of these
experiments was to examine the ability of the phosphatases to dephosphorylate the
Ser245/250/255 linker residues and the SXS motif of Smad2/3. The results showed
that all SCPs could equally dephosphorylate the linker of Smad2/3 in contrast to
PPM1A which did not exhibit such ability. We also demonstrated for the first time
that SCPs can dephosphorylate the SXS motif of Smad2/3 which is phosphorylated by
2
TGF-β receptor type I. Moreover, we confirmed that the MAP kinases JNK and ERK
are responsible for the phosphorylation of the Smad2 linker region.
Consecutively, we investigated the effect of SCP1 on Smad functions such as the
dimerization between Smad3 and Smad4, the binding of Smad3 to the DNA and the
transcriptional activation of reporter genes. The results indicated that the
dephosphorylation of the Smad3 linker region attenuates the dimerization with Smad4
but does not influences the binding to DNA. Furthermore, it was shown a decrease in
the transcriptional activity of Smad2/3 in the presence of even very small amounts of
SCPs, which is in contrast with previous studies.
Finally, we demonstrated that TGF-β does not have an effect on the transcription
of the SCPs and PPM1A genes in HaCaT cells.
|
Ο ρόλος των φωσφατασών SCPI, SCP2, SCP3 ΚΑΙ PPM1A στο σηματοδοτικό μονοπάτι του TGF-b
