(Received for publication, August 14, 1995; and in revised form, October 12, 1995)
From the
The phosphorylation of thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase gene promoters, has been studied. Phosphorylation occurs on a maximum of seven serine residues that are distributed in three tryptic peptides. Mutant derivatives of TTF-1, with alanine residues replacing the serines in the phosphorylation sites, have been constructed and used to assess the functional relevance of TTF-1 phosphorylation. The DNA binding activity of TTF-1 appears to be phosphorylation-independent, as indicated also by the performance of TTF-1 purified from an overexpressing Escherichia coli strain. Transcriptional activation by TTF-1 could require phosphorylation only in specific cell types since in a co-transfection assay in heterologous cells both wild-type and mutant proteins show a similar transcriptional activity.
The thyroid transcription factor-1 (TTF-1) ()is a
homeodomain-containing transcription factor (1) that binds to
the promoters of thyroglobulin (Tg) and thyroperoxidase (TPO) genes,
whose expression is restricted to the thyroid follicular
cells(2, 3) . Transactivation studies demonstrated
that TTF-1 is able to activate transcription from co-transfected
thyroglobulin and thyroperoxidase promoters in non-thyroid cells,
suggesting that TTF-1 may play an important role in the transcriptional
activation of thyroid-specific genes during
development(4, 5) . However, the presence of TTF-1
protein does not always correlate with active transcription of Tg and
TPO genes, as TTF-1 has been demonstrated in tissues other than
thyroid, where no Tg and TPO mRNA could be detected. Furthermore, TTF-1
protein has been detected very early during thyroid development, 5 days
before the appearance of Tg and TPO mRNAs(6) . These data
indicate that, in physiological conditions, TTF-1 is not sufficient to
activate transcription of thyroid-specific genes. Such a notion is
strongly supported by the observation that transgenic mouse lines
carrying a thyroglobulin promoter fused to a chloramphenicol
acetyltransferase gene express the reporter only in thyroid, again
indicating that TTF-1 present in other tissues is unable to activate
the Tg promoter(7) . Interestingly, the promoters of the
surfactant protein B and A genes, exclusively expressed in lung, have
been demonstrated to depend on TTF-1 for
expression(8, 9) . Taken together, these data suggest
that the activity of TTF-1 is differentially regulated.
Phosphorylation is perhaps the most frequent post-translational
modification of those proteins whose activity is regulated in response
to changes in metabolic activity, environmental conditions, and
hormonal signals. Many transcription factors are regulated by
phosphorylation through several distinct mechanisms (10, 11, 12, 13) that can affect
either their DNA binding or their transcriptional activity. We have
previously demonstrated that, in the rat thyroid cell line FRTL-5,
TTF-1 is phosphorylated(14) . Furthermore, in a
Ha--transformed FRTL-5 cells TTF-1 has been
demonstrated to be underphosphorylated and unable to activate
transcription, suggesting that phosphorylation could be an important
mechanism in controlling TTF-1 activity(14) . It has also been
proposed that Ki-ras reduces the capacity of TTF-1 to bind to
DNA via a phosphorylation-dependent mechanism(15) . We report
in this study the mapping of TTF-1 phosphorylation sites. TTF-1 mutants
unable to be phosphorylated show normal levels of DNA binding and
transcriptional activity in heterologous cells, suggesting that
phosphorylation of TTF-1 may have an important role only in specific
cell types.
The amplified products were cloned into the eukaryotic expression vector Rc/CMV (Invitrogen). Finally, restriction fragments containing the serine-alanine substitutions were excised and subcloned in CMV/TTF-1, D14, or D26, described in De Felice et al.(5) , to generate the mutated constructs indicated in Fig. 2and Fig. 4.
Figure 2:
Identification of phosphorylated serine
residues in TTF-1. Panel A, schematic representation of TTF-1
mutants carrying the Ser/Ala substitutions. The constructs were
generated as described under ``Materials and Methods.'' Panel B, HeLa cells transfected with expression vectors
encoding the mutated proteins were in vivo labeled with P, and TTF-1 was purified by immunoprecipitation, then
subjected to electrophoresis, transferred to nitrocellulose, and
subjected to autoradiography (P-32) or Western blot (WB) for quantification. Lanes 1, 2, 3, 4, 5, and 6 are respectively D14, DS40, DS41, DS63, S61, and S80. Panel C, the amino acid
sequence of TTF-1 showing the major phosphorylation sites. The seven
identified phosphoserines are indicated in lower case letters.
The homeodomain is boxed.
Figure 4: Transactivation of the thyroglobulin promoter containing TTF-1 binding sites, by wild-type TTF-1 and various mutant proteins. The indicated amount (expressed in micrograms) of expression vectors encoding TTF-1 either wild type or mutated at the phosphorylation sites were transiently transfected into HeLa cells together with 5 µg of a reporter construct carrying the thyroglobulin promoter fused to the chloramphenicol acetyltransferase gene. The activation values were obtained by dividing the enzymatic activity present in extracts of cells transfected with the various TTF-1 proteins by the activity obtained with the empty expression vector (Rc/CMV).
Figure 1:
TTF-1 is equally
phosphorylated in FRTL-5 cells and upon expression in HeLa cells. Panel A, immunoprecipitation of TTF-1 from FRTL-5- and
HeLa-transfected cells. Left, either thyroid cells (FRTL-5) or HeLa cells transfected with a TTF-1 expression
vector were labeled with [P]orthophosphate.
Equal amounts of TTF-1 were immunoprecipitated from the cell lysates
with specific antibodies, the protein was resolved by a 8% SDS-PAGE and
detected by autoradiography as described under ``Materials and
Methods.'' Lanes 1 and 3 show the phosphorylated
TTF-1 from both cell types. The specificity of the immunoprecipitation
is demonstrated by competition with an excess of the antigenic peptide (lanes 2 and 4). Lanes 5 and 6 show
a Western blot of TTF-1 from FRTL-5 and transfected HeLa cells,
respectively. Panel B, phosphoamino acid analysis of TTF-1
immunoprecipitated from FRTL-5 and transfected HeLa cells (left and right, respectively). Following detection by
autoradiography (see Panel A) the bands corresponding to
P-TTF-1 from FRTL-5 and Hela cells were cut from the gel,
and the protein was processed as described under ``Materials and
Methods.'' The plates were exposed for autoradiography at
-70 °C for 14 days with an intensifying screen. Panel
C, tryptic maps of TTF-1 from FRTL-5- and HeLa-transfected cells (left and right, respectively).
P-TTF-1
was purified by immunoprecipitation and electrophoresis, then eluted
from the gel and subjected to tryptic digestion as described under
``Materials and Methods.'' Tryptic digests (
500
cpm/each) were applied to thin layer cellulose plates, then resolved in
the horizontal dimension by electrophoresis at pH 8.9 (anode to the left) and the vertical dimension by ascending chromatography
as described under ``Materials and Methods.'' The plates were
exposed for autoradiography at -70 °C for 10 days with an
intensifying screen. The arrow marks the site of sample
application.
Figure 3: Phosphorylation does not affect the affinity of TTF-1 for the oligonucleotide C. Purified HeTTF-1 (0.5 ng) (Panel A), purified bTTF-1 (2 ng) (Panel B), HeLa-transfected total extracts (about 3 µg) containing 0.5 ng of wild-type TTF-1 (Panel C), or 0.5 ng of mutant S80 (Panel D), or FRTL-5 nuclear extracts (about 2 µg) containing 0.5 ng of TTF-1 (Panel E) (the concentrations of TTF-1 in the extracts were determined by Western blotting analysis) were incubated for 45 min in binding buffer containing increasing concentrations of labeled oligonucleotide C. Bound and free DNA were visualized by autoradiography, and the data obtained from each titration were plotted in the graphs A-E. HeTTF-1:TTF-1 was purified from overexpressing HeLa cells (37) and bTTF-1:TTF-1 was purified from overexpressing E. coli cells.
Figure 5:
Half-life of TTF-1 protein. FRTL-5 cells
expressing endogenous TTF-1 were in vivo labeled with
[S]methionine in a pulse-chase experiment as
described under ``Materials and Methods.'' After the
labeling, the chase was performed at the different times indicated.
TTF-1 protein was purified by immunoprecipitation, then subjected to
SDS-PAGE and exposed to autoradiography .
Figure 6: TTF-1 can be phosphorylated by protein kinase C in vitro. Bacterially expressed TTF-1 protein was phosphorylated in vitro with protein kinase C. Samples were loaded on a 8% polyacrylamide gel, and phosphorylated proteins were visualized by autographic exposure of the dried gel. To exclude any possible effect from autophosphorylation of the kinase, the experiment was also performed in the absence of the substrate (lane 1).
In this study, we have identified the phosphorylation sites of TTF-1, a transcription factor implicated in the activation of both thyroid- (21, 22) and lung-specific (8, 9) transcriptional units. TTF-1 is phosphorylated exclusively on serine residues in three different tryptic peptides. The number of phosphorylated serines may range from a minimum of 5 to a maximum of 7 sites, given the uncertainty on the cluster of serines at the amino terminus. Fine mapping of the sites has been obtained in HeLa cells, by measuring the incorporation of labeled phosphate into TTF-1 mutants missing putative phosphorylation sites. Interestingly, no sites for protein kinase A are observed, even though an important role has been proposed for phosphorylation of TTF-1 by this protein kinase(15) . The sites that we have identified show homology to CKII (Ser-18), protein kinase C (Ser-4, -23, and -255), or microtubule-associated protein kinase (Ser-328 and -337) phosphorylation sites. These protein kinases are components of signal transduction pathways that have been shown to control thyroid function and to be activated in thyroid cells in response to a variety of stimuli(23) . In our in vitro experiments only protein kinase C was able to phosphorylate TTF-1. This result is of interest since activation of protein kinase C has been suggested to inhibit thyroid cell differentiation(24, 25, 26, 27, 28, 29, 30) . Furthermore, activation of protein kinase C has been implicated in TSH stimulation of thyroid cell growth(31, 32) . Since TTF-1 has been implicated both in the expression of thyroid differentiated function (21) and in the control of thyroid cell growth(33) , it is an attractive hypothesis that some of these functions could be controlled via phosphorylation of TTF-1 by protein kinase C. The data presented in this study do not, at present, support this hypothesis, since we could not demonstrate any alteration in TTF-1 activity as a consequence of the lack of phosphorylation. However, we cannot rule out the possibility that we were unable to provide evidence for the relevance of phosphorylation in TTF-1 because of the transient transfection assay in heterologous cells that we used. The regulation of TTF-1 through phosphorylation may impinge on thyroid-specific mechanisms which do not operate in HeLa cells such as, for example, phosphorylation-dependent interaction (34) with specific co-activators. Future experiments should aim at studying the phosphorylation mutants in thyroid cells. More conclusive are our studies on the role of phosphorylation in the DNA binding activity of TTF-1. Phosphorylation has been indicated as a critical step for the binding of TTF-1 to its target sequence on the Tg promoter (15, 35) as well as on the TSHr one(36) . Moreover, TTF-1 binding was shown to be abrogated when nuclear extracts were incubated with acid phosphatases. Treatment of extracts with protein kinase A (15) was able to restore TTF-1 DNA binding activity, leading to the conclusion that TTF-1 is directly modified by protein kinase A and binds to the Tg promoter only if phosphorylated(35) . In contrast, our results clearly show that phosphorylation does not play an important role in the overall TTF-1 DNA binding activity. This conclusion is based on the comparable affinities toward a double-stranded oligonucleotide containing a well characterized TTF-1 binding site (oligonucleotide C) (17) of wild-type TTF-1, purified from either bacteria or animal cells, and of the S80 mutant, that contains less that 0.1 phosphorus atom/molecule. We cannot exclude that ancillary proteins, which are protein kinase A substrates, could either help or interfere with TTF-1 binding(15) , depending on the physiological conditions. In addition, we have recently discovered that TTF-1 DNA binding activity can be easily lost during extract preparation and can be readily recovered by exposure to reducing agents (37) . It is also conceivable that the exquisite sensitivity of TTF-1 to oxidation could interfere with the interpretation of in vitro treatments with phosphatases and kinases.
Previous data from our laboratory have
demonstrated that TTF-1 is inactive in Ha--transformed
cells, although it is present and capable of binding to
DNA(14) . In this cell line, we have observed a reduced
phosphorylation of TTF-1(14) , and we proposed that this could
be the cause for the inactivity of TTF-1 in transformed cells. In this
respect, it is interesting to note that ras activation has
been reported to affect the stimulation of protein kinase C in Xenopus oocytes (38) and in PC12 cells(39) .
It is also noteworthy the role of Ras proteins in signal transduction
pathways initiated in membrane receptors (insulin-like growth factor-I,
insulin, and epidermal growth factor) and involving
microtubule-activating protein kinases. These alterations in protein
kinases activities can be somehow affecting TTF-1, either by a direct
or an indirect mechanism involving other kinases and/or phosphatases.
The characterization of the phosphorylation sites in TTF-1 may be
instrumental for the elucidation of the mechanisms leading to the
interference between transformation and differentiation in thyroid
cells.