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Phosphorylation cell-specifically modulates the activity of AF-1 of retinoic acid receptor -2
 
Gert E. Folkers, and Paul T. van der Saag
 
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
 
to be submitted in an altered form
 

Abstract

We have previously reported the presence of an autonomous transcriptional activation domain within the N-terminal part of RAR2, that can activate transcription in a cell-type specific fashion. A detailed mutational analysis revealed that this AF resembled the acidic activator VP16. Furthermore putative phosphorylation sites contribute to its activity. These residues are located within the putative amphipatic -helix, which is required for transcriptional activation. We now present evidence that the N-terminal part of the receptor is indeed phosphorylated in vivo. The presence of these phosphorylatable residues clearly contribute to the cell-specific activation by RAR AF-1 since mutation of these residues resulted in a cell type-dependent modulation of AF-1 activity. Furthermore, cotransfection of CKII and PKA expression vectors resulted in a cell type-specific response to overexpression of these kinases. Together these experiments suggest that phosphorylation of phosphorylatable residues modulates the activity of RAR2 AF-1 in a cell-type specific manner.

Introduction

Activation of transcription is thought to be dependent on an interaction between the DNA-bound activator and the general transcription factors. Through stabilization of the pre-initiation complex or by increasing the formation of the pre-initiation complex activators modulate the activity of RNA polymerase II promoters (37). To perform this task, activators interact with one or more proteins which could be a GTF and/or a cofactor/intermediary protein that can communicate with GTFs (37,44). Alternatively activators could be involved in the disturbing or removal of the nucleosomes (46). In the cell DNA is packed in chromatin, arrays of highly ordered nucleosomes, that in term consist of DNA wrapped around histone particles. Packing of DNA in chromatin structure is thought to prevent transcriptional activation. Acetylation of histones results in a change in chromatin structure, thereby allowing binding of activators and/or GTFs, which in turn can promotes transcriptional activation. Therefore activators interact with proteins that are involved in histone acetylation through the histone acetyl transferases or alternatively activators are, in a SWI/SNF/Brahma-complex-dependent manner, remodelling or removing the nucleosomes (46).

Transcription is a highly regulated process, mRNA expression of most genes is regulated tissue-specifically both during embryonal development and in adult live. The mechanism underlying this cell-specificity is largely unknown. The most important aspect for transcriptional activation is the presence of activators (and/or absence of repressors); however also the activator activity is an important aspect in the cell-specific regulation of transcription. Activity can be regulated by the (limiting) amounts of cofactors/intermediary proteins (32). Also the strength of the interaction between the activator and the interacting protein may modulate transcriptional activation cell-specifically. One mechanism for cell-specific regulation of activator activity is protein phosphorylation. The activity of several transcription factors, e.g. CREB (16) and c-jun (39, 33) have been shown to be up-regulated by phosphorylation (for review see 19). Furthermore steroid hormone receptors are phosphorylated in vivo (45), and phosphorylation sites have been mapped (6,10,15). Although the precise function is mostly unknown, a role in modulating transactivation was suggested by phos-phorylation, as has been reported for the vitamin D3 receptor (20) and the estrogen receptor (ER) (1). Also the retinoic acid receptors (RARs) were shown to be phosphor-proteins, as both ligand-dependent and -independent phosphorylation was observed (14,34,35).

RARs belong to the nuclear hormone receptor superfamily which share a common domain structure (11,17,25). The DNA-binding domain which is most conserved among the different members of this family consists of two zinc fingers. The hormone-binding domain is a highly structured region, located at the C-terminal part of the receptor, and is involved in the binding of ligand, dimerization, and hormone-dependent transactivation through an activation function (AF-2), present in the C-terminal part of the LBD. The N-terminal part of the receptor is most variable and has been shown to contain an autonomous activation function (AF-1) (11). We and others have previously reported the presence of two autonomous transcriptional activation functions in RAR (13,29,30) which both activate transcription by distinct, cell-type dependent mechanisms (13). The activation function present in the N-terminal part of the protein (AF-1, formerly called TAF-1), is located in the first 32 amino acids of the receptor, and functions both in the presence and absence of RA (13) Further characterization revealed that AF-1 is unstructured in solution and is dependent on the presence of acidic and hydrophobic amino acids, showing similarities with the viral acidic activator VP16 (12). Furthermore we found that several putative phosphorylation sites, present within this region, are involved in transcriptional activation. Mutation of these phosphorylation sites led to a small (50%) but significant decrease in activity of RAR2 AF-1 in COS cells. (12). The observed cell-specific activity of this activator (13) and the contribution of putative phosphorylation sites in the activity of this activator (12) led us to suggest that phosphorylation could be involved in the cell-specific activation of RAR.

We show here that RAR is phosphorylated at multiple sites, including the AB-region. Transfection of various AF-1 constructs with mutated putative phosphorylation sites revealed that the contribution of these residues to the activity of AF-1 is cell-specific. The involvement of phosphorylation in the cell-specific activity of AF-1 is further confirmed by the cell-specific effect of cotransfection of expression constructs for the kinases CKII and PKA.

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Material and Methods

Plasmids, cell culture and transient transfection.

All plasmids used have been described before (12). Cell culture, transfections transfection, -galactosidase- , CAT- and LUC-assays and Western blotting were performed as described before (12,13).
 

In vitro phosphorylation

Bacterial GST-RAR fusion proteins were purified as described (12). Kinase reactions were performed in 50 mM Tris pH 7.5, 4 mM MgCl2, 0.2 mM EGTA, 0.2 µl [32P]-ATP, 50 µM ATP, 0.05-0.5 µg gluthation-sepharose-bound GST-fusion protein and 0-10 µg nuclear extract (23), or WCE (see above) in 20 µl for 30 min at 30oC. Subsequently GST-fusion proteins were purified by four washes with PBST (PBSø containing 1% (v/v) Triton X100) and separated by SDS-PAGE (12.5%).
 

In vivo labeling and immunoprecipitation

Cells were transfected as described and 12 hrs or 24 hrs post transfection medium was changed for methionine- or phosphate-free DF medium containing 7.5% (v/v) dialyzed serum and cells were labeled with 50 µCi [35S]-methionine/ml (Amersham) or 1 mCi orthophosphate/ml (ICN), for 14 and 4 hrs respectively. 1.0 µM RA was added together with orthophosphate (4 hrs). Cells were washed with cold PBSø (150 mM NaCl, 16 mM Na2HPO4 and 4 mM NaH2PO4), scraped in PBSø and lysed in 40 µl lysis buffer (see western blotting). After centrifugation the cell lysate was precleared in 750 µl IP-buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X100) containing 0.2 mM PMSF and protease inhibitors using 1 µl normal mouse serum and 50 µl 50% (vol/vol) slurry protein A-sepharose in IP-buffer. For phosphate labelling 50 mM NaF, 40 mM -glycerophosphate and 0.2 mM NaVO4 was added to both lysis and IP buffer. The lysates were then incubated for 2 hrs with 75 µl 12CA5 hybrodoma supernatant, thereafter 50 µl 50% (vol/vol) slurry protein A-Sepharose in IP buffer was added and incubated for another hour. Beads were washed 3 times with 1 ml IP buffer, once with 100 mM Tris-HCl pH 7.5, 0.5 M LiCl (1 ml) and once with 10 mM Tris-HCl pH 7.5 (1 ml). Immune complexes were eluted by incubation at 100oC for 5 min in sample buffer. Immunoblotting was performed as described above. Phosphorylation and methionine incorporation was visualized by autoradiography (2-8 days).

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Results and Discussion
 

RAR is phosphorylated in vivo

AF-1 contains relatively many serine/threonine residues which could be putative phosphorylation sites to test this more directly we first constructed a number of GST-fusion proteins containing the AB region or mutants of this region and performed an in vitro phosphorylation assay with nuclear extracts from various cell lines as a source for kinases, as described in Material and Methods. We observed a weak phosphorylation which was both extract- and fusion protein-dependent. The levels of phosphorylation however were not significantly different between extracts of the various cell lines and were not influenced by RA treatment of cell extracts. Furthermore, no correlation was observed between phosphorylation levels in vitro of the various mutants tested and their capacity to activate transcription in transient transfection (data not shown). We therefore decided to investigate the role of phosphorylation in vivo. In order to be able to immunoprecipitate the receptor we cloned a hemagglutinin tag (HA-tag) in front of the receptor and performed immunoprecipitation with extracts of COS cells transfected with epitope-tagged receptor grown in the presence of [35S]-methionine using a monoclonal against this epitope (12CA5). Figure 1A shows that RAR and RAR AB could be specifically immunoprecipitated with the 12CA5 antibody and, migrated according to their molecular mass. Furthermore this labeled protein immuno-reacted with the antibodies 12CA5 or RAR (Figure 1A, ECL; data not shown). Figure 1B shows the result of an immunoprecipitation of COS cells transfected with epitope-tagged RAR, grown for four hours with 32P-orthophosphate, in the presence or absence of RA. A 32P-labeled protein was immunoprecipitated that comigrated with the simultaneously transfected and immunoprecipitated 35S-labelled epitope-tagged RAR. Immunoprobing with RAR-specific antibody confirmed that the labeled proteins were indeed RAR (data not shown). The levels of retinoic acid receptor phosphorylation were not influenced by RA addition to the culture medium for 4 hours, which is in agreement with earlier findings (34). The levels of phosphorylation of other nuclear hormone receptors is sometimes affected by hormonal treatment as is the case for PR (38), GR (31) and ER (1,15), while the RARs (except RAR1 and 3) were all reported to be phosphorylated irrespectively of the presence or absence of ligand (14,34,35).
 

Multiple regions of RAR are phosphorylated

The fact that RAR is phosphorylated (see above and 34) and observations by us (see later) and others (21,36) that the activity of RARs can be upregulated by protein kinase A, suggested to us that phosphorylation may be important for the activity of the receptor. To test this hypothesis an in vivo phosphorylation experiment was performed with COS cells transfected with various hemagglutinin tag-RAR deletion constructs, as indicated in Figure 2A. Immunoprecipitation of these extracts (Figure 2B) showed that the different truncated proteins were all phosphorylated to various levels and these differences were not the consequence of differences in accumulation levels of the various proteins as judged by western blot of the same membranes using the 12CA5 antibody (ECL). The proteins lacking AF-1 (AB) and AF-2 (E) or both ( AB,E) clearly showed phosphorylation, indicating that regions of RAR not directly involved in transactivation also are phosphorylated. Upon deletion of the D region most phosphorylation signal was lost (compare DE with E) indicating that a strong phosphorylation site is located outside a known activation function (AF-1 or AF-2). Deletion of the AB region (AB) however, also resulted in a decrease in phosphorylation levels, as can be seen by comparing the incorporated phosphate in RAR with AB and E with AB,E (taking differences in expression levels of the proteins into account, Fig. 2C). From these experiments we conclude that multiple regions including the N-terminal part of RAR are phosphorylated. The N-terminal part of ER (1,15), GR (6), PR (10) and RAR(35) were reported to be the major site for phosphorylation and these phosphorylation sites were located within the regions required for AF-1 function. Site directed mutagenesis of these phosphorylation sites affected AF-1 activity in case of ER (1,15) while no or only marginal effects were observed for GR (2,26) and PR (41). Here we observed the presence of a major phosphorylation site within the D-region. This region has also been reported to be phosphorylated in PR (10), ER (15) and AR (47), although the functional consequence of this phosphorylation is yet unknown.
 

Involvement of putative phosphorylation sites in cell specific transcriptional activation.

We have previously shown that the AF-1 activity functions cell-specifically, and mutation of putative phosphorylation sites within the AB-region contribute to the activity of AF-1 (12) together with the above presented evidence for in vivo phosphorylation of AF-1, this led us to investigate whether phosphorylation modulates the activity of AF-1 in a cell-specific fashion. We therefore transfected a GAL-responsive reporter construct together with fusion constructs consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the AB-region of RAR or mutants of this region, in which putative phosphorylation sites were altered. If phosphorylation indeed modulates the activity cell-specifically, differences in the contribution of these residues between the various cell lines are expected. As shown in Table 1, mutation of the three critical acidic residues (D3,6,17A) resulted in a complete loss of AF-1 activity in all cell lines similar as reported before (12). Mutation of Ser 9, 11 and Thr 20 to Ala did not significantly decrease activity in P19-EC and COS cells while a 40-60% reduction in activity was found in 293 and T47D cells. The activity of the S22,24,25A mutant was lower than wild-type in all cell lines tested, suggesting that phosphorylation of these residues contributed to transcriptional activation. Mutation of these residues to aspartic acid increased the activity of this activator, suggesting that negative charge, caused by phosphorylation, can be artificially mimicked by introduction of negatively charged residues (Table 1). Similar findings have been reported for ER (1), c-jun (18) and p53 (28). The activity of the Tyr19 mutant was most interestingly. Mutation of this residue had no effect in COS cells, while a decrease in activity was found in 293, T47D, and 3T3 cells and an increase in activity in P19-EC cells. This could mean that phosphorylation of this tyrosine residue leads to a decrease in activity, since upon mutation of this residue to a non-phosphorylateble residue (Ala) an increase in activity is found in those cells that are able to phosphorylate this residue (eg. P19-EC) but not in cells that apparently lack the particular kinase activity. Interestingly, RAR but not RAR1 and RAR1 were reported to be phosphorylated on tyrosine (34), and the tyrosine kinase signal transduction pathway is functional in P19-EC cells (8). To further test the hypothesis that phosphorylation of Tyrosine residue 19 decreases the activity of AF-1, we tested the activity of WT and Y19A in P19-EC and in P19-RPTP, a P19 clonal cell line that overexpresses a receptor phosphor-tyrosine phosphatase (RPTP), which can differentiate into the neuronal direction in the absence of aggregation upon RA-treatment (9). As shown in Figure 3, the activity of WT and mutant GAL-AF1 is similar in the RPTP clone but in the P19-EC cells a two-fold higher activity is found for the Y19A mutant. These results suggest that in the RPTP clone this tyrosine phosphorylation is absent, similar to the situation in COS cells. It is tempting to speculate that Y19 is dephosphorylated directly or indirectly by RPTP in the RPTP overexpressing P19-EC cell line, leading to the loss of negative contribution of phosphorylated Y19 to AF-1 activity. Cell-specific activation has been reported for ER (5,7,42), and interestingly also mutation of identified phosphorylation sites of the mouse ER S118 resulted in a cell type- and promoter-specific decrease in activity (1) as is the case for RAR AF-1 (Table 1, 12), and GR AF-1 (4). This site (S118) was shown to be phosphorylated by MAPK (22), resulting in a two- to three-fold increase in activity of ER AF-1, both in the presence or absence of ligand. This cross-talk between the growth factor signaling pathways and ER emphasizes the role for phosphorylation in cell-specific activation. Various phosphorylation sites for ER were found in vivo by different groups in different cell lines, including the presence of tyrosine phosphorylation (27), while others reported the phosphorylation of Serine residues only (15,1). Furthermore others reported besides the above described Ser118, an additional CKII phosphorylation site within AF-1 in MCF7 cells (3) which was not found by other groups in COS cells (1,15). Together these data are in agreement with the proposed phosphorylation-dependent cell-specific activation of AF-1.
 

Overexpression of PKA and CKII influence AF-1 activity cell specifically.

The above described differences in the contribution of putative phosphorylation sites in the activity of AF-1 suggests that differences in phosphorylation levels are underlying the cell-specific activation by AF-1. Modulation of the activity of certain kinases present in a cell might cell-specifically modulate AF-1 activity. The RA-dependent activity of RARs has been reported to be activated by PKA (21,36) and PKC (40,43). Dephosphorylation of RARs and RXRs by the addition of okadaic acid led to a decrease in activity through a decrease in DNA binding activity of the RAR/RXR heterodimer (24). To investigate whether kinase activity contributes to cell specific RAR2 AF-1 activity we cotransfected expression constructs for protein kinase A (PKA) and casein kinase II (CKII), together with GAL-AF1 and a GAL-responsive reporter in P19-EC cells and in COS cells. Interestingly cotransfection of both kinases caused a repression in P19-EC cells while in COS cell a two- to three-fold activation was observed. These cotransfection experiments suggest that phosphorylation modulate the activity of AF-1, and that differences in phosphorylation between the cell lines is possibly underlying the observed cell specificity.

In conclusion, the autonomous activation function (AF-1) present at the N-terminus of RAR2 activates transcription cell-specifically (13). Here we have presented evidence that putative phosphorylation sites within this AF are involved in this cell specificity (Table 1, Fig. 3). Furthermore the N-terminal part of the receptor containing AF-1, was found to be phosphorylated (Fig 2) and activity was cell-specifically influenced by kinase expression constructs (Fig. 4). This suggests that cell-specific differences in phosphorylation of AF-1 residues are modulating the activity of AF-1 in a cell-specific manner. To directly confirm this hypothesis we are currently mapping the phosphorylation sites of RAR in different cell lines.
 

Acknowledgements

We are grateful to P. Chambon for providing receptor constructs and antisera against RAR. Patricia Swanink for technical assistance, and F.J.M. Vervoordeldonk and J. Heinen for photography. G.E.F is supported by the Dutch Cancer Society.

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