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Cell Growth & Differentiation Vol. 11, 649-654, December 2000
© 2000 American Association for Cancer Research

Articles

Thyroid Transcription Factor 1 Phosphorylation Is Not Required for Protein Kinase A-dependent Transcription of the Thyroglobulin Promoter 1

Antonio Feliciello2, Giovanna Allevato2, Anna Maria Musti, Daniele De Brasi, Adriana Gallo, Vittorio Enrico Avvedimento3 and Max E. Gottesman

Dipartimento di Biologia e Patologia Molecolare e Cellulare, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università "Federico II," 80131 Napoli, Italy [A. F., G. A., A. M. M., D. D. B., A. G., V. E. A.]; Dipartimento Farmacobiologico, Facoltà di Farmacia, Università della Calabria, 87100 Rende (Cosenza), Italy [A. M. M.]; Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro, 88100 Catanzaro, Italy [V. E. A.]; and Institute of Cancer Research, Columbia University, New York, New York 10032 [M. E. G.]

Abstract

Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).

Introduction

Thyroid cells depend on TSH4 for growth and differentiation (1) . TSH acts by binding a seven-loop transmembrane receptor (TSH receptor) and activating a Gs protein, which in turn stimulates adenylyl cyclase (2 , 3) . Most, if not all, the biological effects of TSH are mediated by cAMP (4 , 5) . cAMP binds the regulatory subunits of PKA, which releases the active catalytic subunit (C-PKA; Ref. 6 ). A fraction of the C-PKA migrates to the nucleus and phosphorylates various substrates (7) . Phosphorylation of the CREB induces the formation of active transcriptional complexes on promoters containing a CRE (8) . CREB, however, is not the sole mediator of the cAMP-induced transcription. Thus, the cAMP-PKA pathway stimulates the formation of p300-NFY-promoter complexes by phosphorylating the NFY B subunit (9) . PKA targets CBP/p300 adapters, which are critical components in assembly of the transcription machinery (10) .

The Tg gene is expressed uniquely in differentiated thyroid cells. TSH and cAMP induce Tg transcription both in vivo and in vitro (11 , 12) . The activity of the Tg promoter (pTg) is strictly dependent on TTF1, a nuclear homeodomain protein that binds pTg at discrete sites (13, 14, 15) . The lung-specific expression of the surfactant protein B and A genes is likewise dependent on TTF1 (16 , 17) . TTF-1 is phosphorylated at multiple sites. It has been reported that unmodified TTF1 binds pTg DNA (18) . Furthermore, a nonphosphorylated TTF1 mutant is transcriptionally active in a transient transfection assay (18) . However, the regulation of TTF1 transcriptional activity by PKA and its state of phosphorylation have not been systematically explored. Here we report that PKA is required for TTF1 transcriptional activation of pTg and a synthetic derivative promoter. PKA phosphorylates TTF1 in vivo. However, mutational analysis demonstrates that PKA stimulation of pTg does not require TTF1 phosphorylation.

Results

PKA Stimulates TTF1-dependent Transcription.
To investigate the effects of cAMP-PKA on TTF1 activity, we expressed TTF1 in A126 cells, a derivative of the pheochromocytoma PC12 cell line. A126 is defective in cAMP nuclear signaling and, unlike PC12, does not differentiate in the presence of cAMP. Expression of exogenous C-PKA reverses the block to cAMP-induced transcription and differentiation (19) .

A126 cells were transfected with TTF1 and neomycin resistance genes under the control of the LTRs of RSV (pRSV). Several clones were isolated and characterized for TTF1 expression (see "Materials and Methods"). A pool of clones expressing TTF1 were transiently transfected with a vector that expresses C-PKA, a vector carrying a CAT reporter gene under the control of the Tg promoter (pTg-CAT), a CRE promoter (pCRE-CAT), or the RSV-LTR promoter (pRSV-CAT). Fig. 1ACitation shows that C-PKA is required to activate Tg and CRE promoters, even in the presence of TTF1 or CREB. Induction was not attributable to a global increase of transcription, because the RSV-LTR promoter was equally active with or without cotransfected C-PKA. The transient cotransfection experiments also showed that C-PKA stimulation of pTg-CAT was entirely dependent on TTF1 (Fig. 1B)Citation .



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  		Fig. 1. TTF-1 activation of the Tg and synthetic target promoters is PKA dependent in A126 cells. A, A126 cells were stably transfected with pRSV-TTF1 and pRSV-NEO. A pool of 10 clones expressing TTF1 was analyzed by Western blot analysis, using specific antibodies. Clones expressing TTF1 were further analyzed by transient transfection with pTg-CAT, pCRE-CAT, and C-PKA. B, A126 cells were transiently transfected with pRSV-TTF1, pTg-CAT, and C-PKA expressing vectors. C, A126 cells were transfected with a synthetic TTF1 target promoter (pTgTTF1-CAT) as described in the text with () or without ({blacksquare}) cotransfection with a C-PKA expression vector. The total amount of transfected DNA was adjusted to 40 µg/ml with salmon sperm DNA. A pRSV-LacZ (2.5 µ g/ml) reporter gene was included to normalize for transfection efficiency. CAT activities were quantified by ß-scope and normalized to ß-galactosidase activities. The activity of pRSV-CAT was taken as 100%. Results are expressed as the percentage of enzymatic activity and represent the means of three independent experiments; bars, SD. Stimulation by PKA of TTF1 activity was inhibited by a PKA-specific inhibitor PKI (see "Materials and Methods").

 
These data do not discriminate between direct activation of TTF1 by PKA or activation/inhibition of other transacting factor(s) that might bind to and regulate the Tg promoter. We therefore assayed a synthetic promoter composed of five tandemly arranged head-to-tail TTF1 binding sites located upstream to a TATA box (E1b). The promoter, which depends on TTF1 and presumably no other DNA-binding transacting factor, was fused to a CAT reporter gene (pTgTTF1-CAT; Ref. 20 ).

Fig. 1CCitation shows that pTgTTF1-CAT was efficiently induced by TTF1 and C-PKA as the native Tg promoter. The synthetic promoter, like the Tg promoter, was inactive in the absence of C-PKA. pTgTTF1-CAT was stimulated by TTF1 in a dose-dependent fashion (Fig. 2A)Citation . The decrease in CAT activity at higher TTF1 concentrations appears to result from limiting PKA. Thus, increasing concentrations of the C-PKA vector further stimulated pTgTTF1-CAT at higher TTF1 levels (Fig. 2Citation A, inset). We have also tested C-PKA induction of TTF1 activity in the COS7 cell line, which, unlike A126, efficiently transduces cAMP-PKA signals to the nucleus. Fig. 2BCitation shows that TTF1 alone or in combination with C-PKA stimulated pTgTTF1-CAT in a dose-dependent manner. As expected, the activity of pTgTTF1-CAT in the absence of C-PKA was higher in COS7 compared with A126 cells. Wild-type PC12 cells behaved similarly to COS7 (data not shown). Stimulation by C-PKA was inhibited by contransfection with a vector that expressed a C-PKA-specific inhibitor (PKI) but not by an inactive PKI variant (see "Materials and Methods"). To demonstrate that the basal TTF1 activity in COS7 cells was dependent on constitutive PKA activity, we cotransfected TTF1 with PKI or its inactive mutated version. Table 1Citation shows that inhibition of endogenous basal PKA activity results in down-regulation of TTF1-dependent transcription. Under the same conditions, transcription driven by RSV-CAT or by a Sp1-dependent promoter was not affected by PKI.



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  		Fig. 2. PKA induction of TTF1 transcriptional activity in PKA- and PKA+ cells. Increasing amounts of TTF1 cDNA plasmid were transiently cotransfected with TTF1-CAT reporter cDNA in the presence ({blacksquare}) or absence (•) of 2.5 µg of the C-PKA expression vector in A126 (A) or COS7 (B). Inset in A, CAT activity as function of C-PKA plasmid concentration in the presence of 0.2 µg of the TTF1 expression plasmid. The results represent means of four independent experiments and are expressed as a percentage of CAT enzymatic activity; bars, SD.

 

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  		Table 1 TTF1 transcriptional activity requires PKA

CAT expression driven by several promoter constructs in the presence of absence of PKA specific inhibitor (PKI) or its single-point mutant (PKI mut). +TTF1 + C-PKA indicates transfections, where TTF1 and PKA catalytic subunit expression vectors have been included. Sp1-CAT is a pEMBL8 plasmid containing the SV40 minimal promoter fused to the SP1 binding site derived from the ferritin promoter.

 
Phosphopeptide Mapping Analysis of TTF1.
To determine whether TTF1 is a direct target of PKA, we analyzed the phosphorylation pattern of endogenous TTF1 in differentiated thyroid cells stimulated with TSH or forskolin, which also raises cAMP levels. Cells deprived of TSH for 3 days in a serum-free medium were metabolically labeled with 32[P]Pi for 4 h in the presence or absence of forskolin, a neutral stimulator of adenylyl cyclase. 32P-labeled TTF1 was immunoprecipitated from total cellular extracts, size-fractionated by SDS gel electrophoresis, purified, and digested with the proteolytic enzyme, thermolysin. The resulting peptides were resolved by two-dimensional TLC and detected by autoradiography. Fig. 3Citation shows five major phosphopeptides. Phosphorylation of two peptides was stimulated by forskolin treatment. These peptides are also found in Escherichia coli TTF1 phosphorylated by PKA in vitro (data not shown).



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  		Fig. 3. Effect of cAMP on TTF1 phosphopeptides in FRTL5 thyroid cells. FRTL5 cells were deprived of TSH for 3 days in serum-free medium and metabolically labeled with [32P]Pi for 4 h in the presence or absence of forskolin (40 µM), a neutral stimulator of adenylate cyclase. TTF1 was immunoprecipitated from total cell extracts purified by SDS-PAGE and digested with the proteolytic enzyme, thermolysin. The resulting peptides were size-fractionated by two-dimensional TLC, and the label was detected by autoradiography. Arrows, the peptides a and f that are stimulated by forskolin. O, origin. A representative set of three independent experiments that yielded similar results is shown. An identical phosphopeptide map was observed in cells stimulated with either TSH (10 milliunits/ml) or cAMP analogues.

 
The PKA phosphorylation sites were located by a peptide map analysis of several previously characterized TTF1 deletion mutants (20) . These constructs were expressed in HeLa cells under standard growing conditions. 32P-labeled TTF1 was isolated and digested with thermolysin, and the resulting phosphopeptides were identified as shown in Fig. 4Citation . Fig. 4BCitation shows a schematic diagram illustrating the location of the mutated residues and the corresponding phosphopeptides. The phosphopeptide map of HeLa-expressed TTF1 was similar to that of TTF1 derived from thyroid cells, except for an additional peptide (peptide d, compare Figs. 3Citation and 4ACitation ). Peptide a is located at the COOH terminus and contains serines 328 and 337, whereas peptide f is a subfragment of peptide a that contains serine 337. These data indicate that serine 337 is the target of PKA-induced phosphorylation.



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  		Fig. 4. A, identification of TTF1 phosphorylated peptides. HeLa cells were transiently transfected with expression vectors coding for wild-type TTF1 or various serine/threonine -> alanine mutants (20) . Transfected cells were metabolically labeled for 4 h with [32P]Pi. TTF1 was immunoprecipitated with specific polyclonal antibodies, purified by PAGE, and digested with thermolysin. wt, wild-type TTF1. B, diagram illustrating the location of specific mutations and phosphopeptides. The mutants have alanine substitutions at the following sites: 1, serine 107; 2, serines 4, 12, 18, and 23 (loss of peptide b); 3, serine 255 (loss of peptides e and partly d); 4, serines 4, 12, 18, 23, 107, 255, and 328 (loss of peptides b, d, and e); 5, serines 328 and 337 (loss of peptides a, d, and f); 6, serines 4, 12, 18, 23, 107, 255, and 337 (loss of peptides b, c, d, e, and f); 7, serines 4, 12, 18, 23, 328, and 337 (loss of peptides a, b, c, d, and f). Peptide f is a fragment of peptide a. Mutant 8, serines 4, 12, 18, 23, 255, 328, and 337, is not phosphorylated in vivo (data not shown).

 
Phosphorylation of TTF1 by PKA Is Not Required for PKA Induction of Its Transcriptional Activity.
TTF1 mutants carrying alanine substitutions for serines 328 and 337 (mut.5) or serines 4, 12, 18, and 23 (mut.2) were tested in A126 for induction of pTgTTF1-CAT in the presence or absence of C-PKA. Surprisingly, no differences in C-PKA-induced activity between wild-type and TTF1 mutants were detected (Fig. 5)Citation . Because TTF1 threonine (9) is essential for PKA-dependent transcription of the surfactant B gene promoter (16) , we tested a mutant carrying a threonine (9) -> alanine substitution in the same assay. Fig. 5Citation shows that the activity of this mutant (mut.9) and of a mutant carrying substitution of serine 337 -> to alanine (mut.6) was significantly stimulated by PKA at both pTg and pTgTTF1. To confirm that TTF1 phosphorylation is not required for C-PKA-induced activity, we tested a mutant lacking all phosphorylation sites (mut.8, see the legend of Fig. 4Citation ). Similar to wild-type TTF1, mut.8 activates the transcription of the target promoters only in the presence of C-PKA (Fig. 5)Citation .



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  		Fig. 5. The activity of the TTF1 mutants still depends on PKA. Wild-type TTF1 or the mutants described in Fig. 4Citation were coexpressed in A126 cells with pTgTTF1-CAT in the presence () or absence ({blacksquare}) of PKA expression vectors. Mutant threonine (9) -> alanine alters a TTF1 residue reported to be phosphorylated by PKA in lung (16) . The results are expressed as CAT enzymatic activity and represent means of four independent experiments that yielded similar results; bars, SD.

 
These data indicate that PKA phosphorylation of TTF1 is not required for induction of TTF1-dependent transcription. This implies that C-PKA phosphorylates and activates or inhibits other factor(s), possibly not thyroid specific, that regulates TTF1-mediated transcription.

Discussion

TTF1 transacting factor is expressed exclusively in thyroid and lung and appears to be necessary for the correct development of these organs (21) . We showed that PKA signaling up-regulates the transcriptional activity of TTF1-dependent promoters. This finding explains a well-established phenotype, i.e., TSH induction of thyroid-specific genes in vivo and in vitro (11 , 12 , 22) . It has been shown previously that transformation by Ras oncogenes represses the expression of TSH and TTF1-regulated genes (23, 24, 25, 26) . Furthermore, we demonstrated that Ki-Ras oncogene interferes with differentiation of thyroid cells by down-regulating nuclear C-PKA signaling (27, 28, 29, 30) . The data shown above link PKA to TTF1; PKA is required for TTF1-regulated transcription.

The extent of stimulation of TTF1-dependent transcription by PKA depends on cell type. In cells with a wild-type PKA background (COS7 and PC12), the fold stimulation by PKA is slight (2-fold), whereas in PKA-deficient cells (A126), the fold stimulation is considerably higher (7–8-fold). The data in Table 1Citation indicate that TTF1-dependent transcription is sensitive to basal levels of PKA activity and implies that thyroid-specific gene expression is controlled by TSH at physiological levels (11) .

To elucidate the mechanism of action of cAMP in the expression of thyroid-specific genes, we analyzed the phosphorylation pattern of TTF1. We showed that phosphorylation of TTF1 residue serine 337 was stimulated by PKA (Fig. 4)Citation . However, substitution of this serine with alanine did not affect PKA-dependent transactivation (Fig. 5)Citation . Furthermore, unphosphorylated TTF1 (mut.8) still stimulated transcription in a PKA-dependent fashion (Fig. 5)Citation .

Residue threonine (9) at the NH2 terminus of TTF1 was revealed as a PKA phosphorylation target in lung cells, and its modification was shown to be necessary for PKA-induced transcription of the surfactant B gene (16 , 17) . This residue is not phosphorylated in thyroid cells, and furthermore, TTF1 bearing an alanine substitution at this position (mut.9) activated the thyroglobulin and synthetic promoters in a PKA-dependent fashion. This discrepancy may indicate that TTF1 phosphorylation may be essential in some tissues and not in others. Alternatively, it may reflect some difference in the experimental protocol unrelated to physiological mechanisms.

We have shown previously that in thyroid cells, TTF1 binding activity was stimulated by exogenous or endogenous cAMP-PKA (17 , 27 , 30, 31, 32) . On the other hand, the binding of recombinant wild-type or mutant TTF1 to the Tg promoter does not require PKA phosphorylation (18) .5

We propose that transactivation by TTF1 depends on some other factor(s) phosphorylated by PKA. This factor(s) cannot be thyroid specific because PKA stimulates transcription of TTF1-dependent promoters in several nonthyroid cell types (PC12, COS7, and HeLa). In addition, the factor may not bind DNA at a TTF1 promoter, because PKA stimulates a synthetic promoter, the enhancer of which contains only TTF1 binding sequence repeats. The putative factor might be either a PKA-dependent activator or a repressor that is suppressed by PKA.

Materials and Methods

Cell Lines.
FRTL5 cells are a continuous line of functional epithelial cells derived from normal rat thyroid (33) . They are cultured in Coon’s modified F-12 medium supplemented with 5% calf serum (Life Technologies, Inc.), plus six hormones: 1 nM TSH, 10 µg/ml insulin, 10 nM hydrocortisone, 5 µg/ml transferrin, 10 ng/ml glycyl-L-histidyl-L-lysine acetate, 10 ng/ml somatostatin (33) . HeLa and COS7 cells were grown in DMEM supplemented with 10% FCS (Life Technologies, Inc.). A126–1B2 cells were grown in RPMI 1640 containing 5% FCS, 10% horse serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine as described (19) . For transient expression assays of TTF1, cells were plated at 1 x 106 in a 100-mm-diameter tissue culture dish 1 day before transfection.

DNA Plasmid and Transfections.
Cells were transfected using calcium phosphate (34) . For transactivation experiments, 2 x 106 cells were plated 12 h before transfection. Plasmid cDNAs were used at the indicated concentrations. cDNA for wild-type or several deletion mutants of TTFl were subcloned into constitutive expression vector pRc/RSV (Invitrogen). pTg-CAT carries 830 bp of pTg (-827 to +3 from the transcription start site) fused to the CAT gene (35) . The pCRE-CAT fusion carries the CRE element from the c-fos promoter fused to the TK-CAT reporter gene (36) . pRSV-C-PKA was kindly provided by Dr. S. McKnight (University of Washington, Seattle, WA). Cell extracts were prepared 48 h after transfection. CAT and ß-galactosidase activities were determined as described (34 , 35) . All experiments involving transfections with C-PKA included DNA expression vectors carrying the PKA specific inhibitor, PKI, or its inactive mutated version as controls (37) . A126 cells expressing TTF1 were isolated after transfection with pRSV-TTF1 (5 µg) and a pRSV-neomycin (aminoglycoside phosphotransferase) resistance gene (2 µg). Neomycin-resistant clones were isolated and characterized for TTF1 expression by immunoblot analysis. Ten positive clones were pooled and used for transient transfection with C-PKA.

In Vivo 32P-Labeling and Immunoprecipitation Experiments.
FRTL5 cells were grown to semiconfluence in the presence of serum and hormones, starved for 3 days in 0.1% BSA, and metabolically labeled with [32P]Pi in the presence or absence of 40 µM forskolin for 4 h. HeLa cells were transiently transfected with expression vectors coding for wild-type TTF1 or mutants in which various serine residues were substituted with alanine. Transfected cells were metabolically labeled for 4 h in phosphate-free medium with inorganic [32P]Pi. 32P-labeled TTF1 was immunoprecipitated from total cell extracts with specific polyclonal antibodies, resolved on SDS-PAGE, and purified from the gel. Purified 32P-labeled TTF1 was subjected to proteolytic digestion with thermolysin (Sigma Chemical Co.) and analyzed by two-dimensional TLC as described (38) .

Acknowledgments

We thank Dr. R. Di Lauro and his co-workers for providing us with all of the materials necessary for studying TTF1 phosphorylation: TTF1 antibody, E. coli-TTF1, and HeLa-TTF1. Special thanks to Dr. S. Zannini for helpful discussion.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro; Targeted Project Biotechnology Consiglio Nazionale delle Ricerche; Ministero dell’Universitá e Ricerca Scientifica e Tecnologia (Italian Department of University and Research); the Lucille P. Markey Charitable Trust, Grant POl CA23767-15 from the NIH; and the Associazione Italiana Ricerca Cancro. A. F. was supported by the Associazione L. Di Capua and Associazione Italiana per la Ricerca sul Cancro Fellowships. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Dipartimento di Biologia e Patologia Molecolare e Cellulare, Facoltà di Medicina, II Policlinico via S. Pansini 5, 80131, Napoli, Italy. Phone: 39-81-7463251; Fax: 39-81-7463252. Back

4 The abbreviations used are: TSH, thyroid stimulating hormone; cAMP, cyclic AMP; PKA, protein kinase A (cAMP-dependent protein kinase); C-PKA, catalytic subunit of PKA; TTF1, thyroid transcription factor 1; CREB, cAMP response element binding protein; Tg, thyroglobulin; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyltransferase; LTR, long terminal repeat. Back

5 A. M. Musti, unpublished observations. Back

Received for publication 7/ 3/00. Revision received 10/12/00. Accepted for publication 10/17/00.

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