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Environmental Toxicology Graduate Program (K.S.), Department of Cell Biology and Neuroscience (V.M., V.P., F.M.S.), Cell, Molecular and Developmental Biology Graduate Program (K.C.), University of California, Riverside, California 92521; Center for Cell Signaling (B.M.P.), Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908; Département de Biologie et Génomique Structurales (Y.B., D.M.), Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, France; and Division of Pulmonary Biology (Y.M.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229
Address all correspondence and requests for reprints to: Dr. Frances M. Sladek, Department of Cell Biology and Neuroscience, 2115 Biological Sciences Building, University of California, Riverside, California 92521-0314. E-mail: frances.sladek{at}ucr.edu.
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ABSTRACT |
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(HNF4). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4 and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4 (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4 as well as decreased endogenous HNF4 protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor , retinoid X receptor , and thyroid hormone receptor ß) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor and peroxisome proliferator-activated receptor . Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs. |
INTRODUCTION |
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Phosphorylation is an important ligand-independent mechanism that regulates essentially every aspect of NR function, just as it does that of other transcription factor families (21, 22, 23, 24). NRs are phosphorylated in their transactivation domains [the activation function 1 (AF-1) region and the LBD] and the DBD (reviewed in Refs. 1 and 22). Phosphorylation of the LBD and AF-1 by different kinases may affect their dimerization (25, 26, 27), cofactor recruitment, and transactivation (28, 29, 30, 31, 32, 33). Phosphorylation of the DBD has also been shown to impair receptor dimerization and DNA binding activity of several NRs (22, 34, 35, 36, 37, 38). Because several NR nuclear localization (NLS) and export signals (NES) are located in the DBD, it is possible that phosphorylation of the DBD will also affect the nuclear localization of NRs, as it does that of other transcription factor families (e.g. Refs. 6 and 39, 40, 41, 42, 43).
Hepatocyte nuclear factor 4 (HNF4; NR2A1) is a highly conserved, constitutively active NR that binds DNA exclusively as a homodimer and is implicated in several human diseases, including maturity onset diabetes of the young 1 (MODY1), hemophilia, and atherosclerosis (reviewed in Ref. 44). HNF4 exists primarily in the nucleus (45), but considering its constitutive activity, and the possibility of binding a ubiquitous ligand (46, 47), it is reasonable to anticipate that there are other, as-yet-unidentified, mechanisms by which HNF4 activity is regulated. For example, CREB-binding protein-mediated acetylation appears to be required for the proper nuclear retention of HNF4 (48). HNF41 has been previously shown to be phosphorylated in as many 13 sites (Ref. 49 ; and Sladek, F. M., L. Nepomuceno, J. Evans, J. Michels, and A. Burlingame, unpublished data), although only a few phosphorylation sites have been mapped. Protein kinase A (PKA) phosphorylates HNF41 at serine (Ser, S)134 in the DBD and impairs its DNA binding ability (36). AMPK phosphorylates HNF41 at S304 in the LBD and impairs its dimerization and DNA binding activity (Ref. 27 ; and Sun, K., K. Zhang, Y. Brelivet, D. Moras, and F. M. Sladek, manuscript in preparation). p38 kinase phosphorylates S158 in helix 2 of the LBD of HNF41 in response to inflammatory redox, causing an increase in DNA binding and transactivation (50). However, the function of the phosphorylation of other sites and the physiological and pathological pathways that lead to phosphorylation have not been well characterized. Furthermore, HNF4 is considered to be one of the more ancient NRs (51) and the HNF4 gene family is expanded approximately 250-fold in nematodes (52), so that findings for mammalian HNF4 may be relevant to other NRs as well.
Protein kinase C (PKC) comprises a large family of Ser/threonine (Thr) kinases that are activated by many extracellular and intracellular stimuli. They are categorized into three subfamilies on the basis of their structure and ability to bind diacylglycerol and calcium ions (Ca2+): classical (, ßI, ßII, 
), novel (
, 
, 
, 
), and atypical PKC (
, 
) (53). PKC plays a fundamental signaling role in many physiological processes, including modulating membrane structure, mediating the immune response, and regulating cell proliferation and differentiation via phosphorylation of various transcription factors (54, 55). Generally, inactive PKCs are considered to be primarily cytoplasmic. However, upon activation by different signals, PKC may translocate to the nucleus (54). The nuclear PKCs have been shown to phosphorylate and regulate the activity of several NRs. For example, human RAR can be phosphorylated by PKC resulting in a decrease in DNA binding activity, dimerization, and transactivation (37); vitamin D receptor (VDR) DNA binding and transactivation is inhibited by PKC phosphorylation (35, 56); and peroxisome proliferator-activated receptor (PPAR) DNA binding, heterodimerization with retinoid X receptor (RXR), and transactivation/repression are all affected by PKC phosphorylation (38, 57). More recently, treatment by PKC activator phorbol 12-myristate 13-acetate (TPA) was shown to induce mitochondrial localization of orphan receptor Nurr77 and RXR (58), as well as to promote the degradation of RXR (59).
In the current study, we shed additional light on the role of PKC phosphorylation in NR function and propose a mechanism by which NRs may be degraded or repositioned to exert nongenomic effects. Specifically, we show that HNF4 is phosphorylated by PKC in vitro at S78 between the two zinc fingers and that a phosphomimetic mutant of that site (S78D) impairs DNA binding and transactivation and facilitates degradation by the 26S proteasome pathway. It also increases cytoplasmic localization, an effect that is greatly enhanced in the MODY1 Q268X mutant and that could be due to neutralization of positive charges adjacent to S78 that we also show play a role in nuclear import. Finally, because S78 and the positively charged residues arginine (Arg, R) and lysine (Lys, K) adjacent to S78 are highly conserved in most mammalian nonsteroid NRs, we investigated the role of ‘S78’ phosphorylation in other NRs and found that the DBD of RXR, RAR, and TRß are all phosphorylated by PKC in vitro. We also found that phosphomimetic mutants of ‘S78’ in PPAR and RXR lead to cytoplasmic localization. Therefore, we propose that phosphorylation of ‘S78’ in the NR DBD may be a common trigger for turning off NR genomic function, and, for certain NRs, turning on nongenomic functions.
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). Of the 46 human NRs with a DBD, all but five receptors have a Ser or Thr at that position. The exceptions—GR, AR, PR, SXR, and mineralocorticoid receptor (MR)—have an alanine (Ala) at that position and all bind steroids (alignment on the left). The only classical steroid receptors that contain a Ser or Thr at that position are the estrogen receptors (ER and ERß). Several positively charged Lys or Arg residues close to the Ser/Thr are also highly conserved. In HNF41, the relevant residues are F74, F75, R76, R77, S78, R80, and K81. The sequence content of S78 in HNF41 suggests it is a PKC phosphorylation site (Fig. 1B). The sequence of the other NR DBDs with a Ser or Thr at that position also match the PKC consensus but to varying degrees. This region of the NR DBD has been shown to be important for nuclear localization of RXR, VDR, SXR, and the Rev-erb receptors (60, 61, 62, 63, 64). In addition, the conserved serine has been shown to be a target of PKC phosphorylation in VDR (S51) and PPAR (T129) (35, 38).
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in Vitro on S78, a recombinant His-tagged HNF41 was incubated with a mixture of PKC isoforms (, ß, ) in the presence of [-32P]ATP. HNF41 was phosphorylated by PKC in vitro, and the phosphorylation was efficiently inhibited by a PKC peptide inhibitor (Fig. 2A). To determine whether S78 is phosphorylated by PKC, a S78A mutation was introduced into a glutathione S-transferase (GST) fusion construct containing the HNF4 DBD (GST.DBD.S78A). The results show that PKC phosphorylates GST.DBD.wt well (Fig. 2B, lane 2) but fails to phosphorylate the GST.DBD.S78A mutant (lane 3). Thus, PKC phosphorylates the HNF4 DBD, and under these conditions, S78 is the major phosphorylation site. We also found that PKC phosphorylates S78 in the context of the full-length protein (His.HNF41) by showing a decrease in phosphorylation in a double mutant of S78A and S304A (Fig. 2C, compare lane 3 with lane 2); S304 is another PKC target site (Sun, K., K. Zhang, Y. Brelivet, D. Moras, and F. M. Sladek, manuscript in preparation).
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Impairs DNA Binding and Transactivation function, a transient cotransfection assay was carried out using an HNF4-responsive promoter linked to luciferase. The HNF4 phosphomimetic mutant S78D exhibited a greatly decreased ability to activate transcription, whereas the S78A mutant showed much less of a decrease in activity (Fig. 3A). Because phosphorylation of S78 introduces a negative charge in the DBD, we next investigated whether S78D would affect DNA binding. The results showed that the S78D mutant of HNF41 expressed in COS-7 cells fails to bind an HNF4 response element in a gel shift assay (Fig. 3B, lower panel, lane 3). In contrast, neither the S78A mutant nor other Ser to aspartic acid (Asp, D) mutations in the HNF4 DBD (S86D, S90D) showed an appreciable decrease in DNA binding activity (lanes 2, 5, and 7). Immunoblot analysis verified that similar amounts of the wild-type (wt) and mutant of proteins were used in the gel shift assay (Fig. 3B, upper panel). To determine whether HNF4 responds to PKC signaling pathways in vivo, HepG2 cells, a human hepatoblastoma/hepatocellular carcinoma cell line that expresses endogenous HNF4, were treated with the PKC activator TPA. Gel shift analysis of nuclear extracts from these cells also showed a decrease in HNF4 DNA binding, suggesting that PKC affects the DNA binding ability of the endogenous HNF4 protein (Fig. 3C).
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DBD on the DNA in the absence and presence of a phosphate group at S78 (Fig. 3D
). The threaded structure shows that phosphorylation of S78 requires a conformational change in order for the kinase to reach the Ser, implying that there are dynamic fluctuations in the HNF4 DBD. There are two major orientations for the phosphorylated Ser, both of which could explain a lack of DNA binding. In the orientation shown, the phosphoryl group points toward a hydrophobic cluster consisting of F74, F75, F112 leucine (Leu, L)108 and destabilizes it. As a consequence, the structure would be altered with a relative displacement of helices 2 and 3, which in turn would affect DNA binding (i.e. via R125). In the other orientation (data not shown), the phosphorylated Ser points toward the DNA, a position that can be obtained only by a conformational change of the loop connecting helix 1 and helix 2, most likely through a melting of the C-terminal end of helix 1. It is anticipated that either of these structural changes in that region of the DBD would also affect the numerous contacts with the DNA by the Arg residues, as indicated by comparison with the crystallographic structure of the DBD of RXR, the NR most similar to HNF4 (65).
HNF4 Protein Levels Are Down-Regulated by PKC Phosphorylation of S78
To further investigate the effect of PKC signaling pathways on HNF4 in vivo, we examined the levels of endogenous HNF4 protein in TPA-treated HepG2 cells. The results show a time-dependent decrease in the amount of HNF4 protein in the presence of TPA that is blocked by the addition of bisindolylmaleimide II (BisII), a specific inhibitor of PKC (Fig. 4A, compare lanes 6 and 7 with lanes 10 and 11). Because HNF4 mRNA levels were not affected by a similar TPA treatment (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), this suggests that TPA affects the level of the HNF4 protein. Furthermore, because the TPA-mediated reduction of the HNF4 protein is inhibited by MG132, we infer that PKC signaling promotes HNF4 turnover via the proteasome pathway (Fig. 4B, compare lane 6 with lane 3).
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protein levels, the stability of HNF4 wt and phosphomimetic mutants S78D and S304D was examined in COS-7 cells. The results show that the S78D protein disappears at an earlier time point than the S304D protein, which exhibited a stability similar to that of the wt protein (Fig. 4C, compare lane 9 with lanes 15 and 3, respectively). S78A (and S304A) also exhibited a half-life similar to that of wt HNF4 (
12 vs. 6 h for S78D, lanes 3, 6, and 12), suggesting that the Asp (D) at residue 78 is responsible for the shorter half-life and not some other structural change introduced by the mutation. Activation of PKC by TPA treatment in COS-7 cells also leads to faster degradation of exogenously expressed HNF4 wt protein, but has little effect on the S78A mutant (Fig. 4D, compare lane 6 with lane 3, and lane 12 with lane 9). These results strongly suggest that S78 is a target of PKC signaling in vivo, and that S78 phosphorylation plays a role in the down-regulation of HNF4 protein by PKC.
Phosphomimetic Mutant S78D Exhibits Some Cytoplasmic Localization
We next determined the intracellular localization of the S78D mutant. Green fluorescent protein (GFP)-fused HNF4 wt and S78D were expressed in COS-7 cells in the presence of MG132 to stabilize the protein, immunostained with affinity-purified antisera specific to HNF4 (445), and imaged by confocal microscopy. The results show that the GFP-fused HNF4 wt is exclusively in the nucleus, as we have seen previously (45). In contrast, the GFP-fused S78D mutant showed some localization in the cytoplasm, in addition to the nucleus (supplemental Fig. S2A), as did the nonfusion protein of S78E, the other phosphomimetic mutant (supplemental Fig. S2B). (See Fig. 5B for cytoplasmic localization of the nonfusion S78D protein; it is less than that of the GFP fusion protein, which may be due to enhanced stabilization by the GFP moiety.)
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) and are part of the NLS in RXR and VDR (60, 61). We found that mutations in these residues (RR76,77GW and RK80,81GW) (Fig. 5A) also result in enhanced cytoplasmic localization of HNF4 (Fig. 5B, top panels), which is not altered by the addition of leptomycin B (LMB), an inhibitor of chromosome region maintenance 1 (CRM1) nuclear exporter (Fig. 5B, lower panels). The S78D mutant also exhibited cytoplasmic localization, although less than the double mutants, and it was similarly not affected by LMB treatment (Fig. 5B). [An additional NLS has been mapped in HNF4 in the hinge region (48), which could account for the remaining nuclear localization of S78D.] The cytoplasmic localization is evident in the majority of cells harboring the mutants (for additional images, see supplemental Fig. S3) and was quantified by determining the cytoplasmic to nuclear ratio of HNF4 wt and the three mutants (Fig. 5C). Enhanced cytoplasmic localization was also observed (and quantified) in the TPA-treated HepG2 cells (Fig. 5D), lending physiological relevance to the finding.
A second nuclear export pathway involving the DBD of NRs has been reported; it involves the two highly conserved Phe residues (F74 and F75 in HNF41) (see Fig. 1A) adjacent to S78 and recognition of the DBD by the chaperone calreticulin (CRT) (39, 66, 67). To determine whether phosphorylation of S78 affects this export pathway, the wt and S78D HNF4 were transfected into CRT–/– mouse embryonic fibroblasts. Immunofluorescence microscopy showed that the nuclear and cytoplasmic levels of the S78D mutant were comparable in the CRT+/+ and CRT–/– cells (supplemental Fig. S4). This suggests that CRT does not facilitate the cytoplasmic localization of HNF4 that is regulated by S78 phosphorylation. Taken together, these results suggest that the positively charged residues proximal to S78 may be involved in nuclear import and/or retention of HNF4, and introduction of a negative charge, such as a phosphate group, in that region may adversely affect the localization.
The MODY1 Mutant Q268X Enhances the Cytoplasmic Localization of the S78D Mutation in HNF4
Because the LBD of NRs, and the AF-2 in particular, are known to play a role in receptor turnover (15), we investigated the effect of introducing S78D into a naturally occurring mutation in HNF4 found in MODY1 patients (Q268X), which results in a truncated LBD (68). The results show that the S78D mutation had a more profound effect on the localization of the MODY1 mutant than it did on the full-length HNF4 (Fig. 5, compare E with B). Somewhat uneven staining in the nucleus of the wt Q268X turned to very prominent focal staining in the nucleus as well as the cytoplasm in the Q268X S78D double mutant. These results further support the notion that S78D influences the intracellular localization of HNF4 and provide an example of how a signal transduction pathway may differentially affect wt and mutant proteins.
Phosphomimetic Mutations of the Residue Analogous to S78 Induces Cytoplasmic Localization of Other NRs
Because the amino acid corresponding to S78 in HNF41 is a Ser or Thr in 41 of 46 human NRs (Fig. 1A), we examined whether the Ser/Thr in select other NRs is also phosphorylated by PKC and whether the phosphorylation regulates subcellular localization. Recombinant GST.hRAR.DBD, GST.hRXR.DBD, GST.hTRß.DBD incubated with the PKC isoform (, ß, ) mixture and [-32P]ATP all exhibited specific phosphorylation by PKC (Fig. 6A). Furthermore, a ‘S78D’ mutant of GST.hRXR.DBD (T162D) was not phosphorylated by PKC (Fig. 6B), suggesting that ‘S78’ is the only PKC target in the human RXR DBD. Confocal microscopy showed that the ‘S78D’ phosphomimetic mutant of a GFP.RXR construct (T162D) is located primarily in the cytoplasm (Fig. 6C). This was true for both a C-terminally truncated RXR [GFP.RXR.
C, amino acids (aa) 1–235] (left panels) as well as a full-length RXR (right panels). Interestingly, the full-length ‘S78D’ construct displayed nuclear staining that was not observed with the ‘S78D’ truncated version, which could be due to the presence of an additional NLS in the RXR LBD (58). Costaining for the mitochondrial heat shock protein 70 (mtHSP70) suggests that there might be some localization of the ‘S78D’ RXR mutant in the mitochondria (Fig. 6C, merge panels) (see below for further discussion). Because PPAR has been shown by others to be phosphorylated by PKC on ‘S78’ (Thr129) (38), we made the corresponding phosphomimetic mutant of full-length PPAR (T129D) fused to GFP and found that it too is localized to the cytoplasm, whereas the wt receptor is found solely in the nucleus (Fig. 6D). Taken together, these results support the notion that phosphorylation of ‘S78’ in the DBD of NRs may be a common mechanism that regulates the subcellular localization of NRs. (For additional images at low magnification, showing the effect in >50% of the transfected cells, see supplemental Fig. S5.)
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DISCUSSION |
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can be phosphorylated by PKC in vitro at S78 in the DBD and that the phosphomimetic mutant S78D fails to bind DNA and activate transcription (Figs. 2 and 3). Modeling of the HNF4 DBD provides a structural basis for these results (Fig. 3D). We also show that PKC stimulates degradation of the HNF4 protein via the proteasome pathway and that an S78A mutant is resistant to that degradation (Fig. 4). A phosphomimetic mutant S78D results in partial cytoplasmic localization, which is analogous to that induced by mutations in nearby positively charged residues (R76, R77, R80, and K81) (Fig. 5 and supplemental Figs. S2 and S3). The physiological relevance of these findings was reinforced with studies using TPA-treated HepG2 cells, as well as a naturally occurring MODY1 mutation (Figs. 3–5). Finally, we show that other NR DBDs are also phosphorylated by PKC (RAR, RXR, TRß), and that a phosphomimetic mutation at ‘S78’ results in nonnuclear localization of two receptors (RXR, PPAR) (Fig. 6 and supplemental Fig. S5). Because ‘S78’ is conserved in most NRs (Fig. 1), we propose that phosphorylation of ‘S78’ may be a common mechanism that regulates the function of these NRs (see Fig. 7).
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induced by PKC may involve interference with the positively charged residues (R76, R77, R80, and K81) that surround S78, which comprise an NLS. This region has also been shown to be an NLS for other NRs (60, 61, 62, 63, 64), although until now there has not been a detailed examination of the effect of PKC phosphorylation of ‘S78’ on nuclear localization. Here, we show that ‘S78’ (T162) in the RXR DBD is phosphorylated by PKC and that introduction of a single negative charge at that position results in a remarkable cytoplasmic localization. RXR has been shown previously to heterodimerize with other NRs, and upon treatment of TPA to localize to the mitochondria (58). Here, we observe the majority of the mutant RXR in the same region of the cell as the mitochondria but only a little colocalization with mtHSP70 (yellow color in the merge panel), suggesting that most of the ‘S78D’ mutant may not be actually in the mitochondria. These findings are reminiscent of those found by others in which a naturally occurring mutation in the AR DBD results in localization proximal to but not in the mitochondria (69). Others have shown that TPA also induces the degradation of RXR through the proteasome in gastric cancer cells (59), a finding reminiscent of the increased degradation we observe for HNF4.
We show that a single phosphomimetic mutation of ‘S78’ in PPAR (T129D) also results in a strong cytoplasmic localization in at least 50% of the cells. Others have shown previously that PPAR is phosphorylated by PKC in vitro and in vivo at T129 and observed an inhibition in DNA binding in a T129D mutant, reminiscent of our findings with HNF4; but they did not examine the effect of the phosphomimetic mutant on intracellular localization (38). Whereas several other NRs have been reported to be localized to the mitochondria under different conditions (GR, ERß, and TR) (6), we did not observe any costaining of the ‘S78D’ PPAR mutant with mtHSP70. VDR has also been shown previously to be phosphorylated on ‘S78’ (S51) by PKC, resulting in an inhibition of DNA binding and transactivation; preliminary data not shown by this group also suggested that phosphorylation of S51 may affect nuclear localization (35, 56). Finally, we also show here that the TRß and RAR DBD are both phosphorylated by PKC in vitro. The target sites have not yet been mapped, but both receptors contain the conserved Ser at position 78 surrounded by positively charged residues that constitute a PKC consensus site (Fig. 1) and both TR (8) and RAR have been shown to exhibit cytoplasmic localization under different conditions, including TPA treatment (70).
Finally, a comment about receptor solubility and the use of mutant receptors to study protein function is warranted. Because mutations in other proteins are known to cause misfolding and result in insoluble protein aggregates (e.g. Refs. 71, 72, 73) and because in some of our images we observed what appeared to be aggregates of the receptors (Figs. 5 and 6), we examined the solubility of the receptors and the ‘S78D’ mutants in the transfected COS-7 cells. The results show that the majority of both the wt and mutant receptors were in the soluble fraction of the cell; only PPAR was present to an appreciable degree in the insoluble fraction, but the amount did not increase in the ‘S78D’ mutant (data not shown). Furthermore, because PKC is known to phosphorylate NRs on multiple sites, to study the effect of a single phosphosite point mutations have to be used. Phosphomimetic mutations of Asp (D) and Glu (E) are widely used under such situations, but it must be kept in mind that they may not completely recapitulate the effect of an added phospho group and that multiple sites might be involved in vivo. Additional studies are required to determine the function, if any, of these phosphomometic mutants (and by extension PKC-phosphorylated NRs) in the cytoplasm.
With these caveats in mind, the results presented here, along with the work by others, lead us to propose a potential common mechanism by which the majority of NRs may be affected by PKC signaling—namely, phosphorylation of the conserved Ser between the two zinc fingers, resulting in decreased DNA binding and altered intracellular localization (Fig. 7). What apparently differs among the NRs is the ultimate fate of the receptor once outside of the nucleus. In the case of HNF4, it appears to be degradation by the proteasome, whereas in the case of RXR and PPAR it appears to be localization to different regions of the cytoplasm with some potential mitochondrial localization for RXR. In either case, phosphorylation by PKC results in a decrease in NR genomic function and a potential increase in nongenomic effects.
Our findings also indicate that PKC signaling may result in different outcomes for wt and naturally occurring mutants, which could play a role in the pathology of some diseases. When MODY1 was first linked to mutations in HNF4, it was noted that not all family members with the Q268X mutation had diabetes (68). The reason for this is not known, but the findings presented here suggest one potential explanation. We observed that S78D resulted in more prominent cytoplasmic staining in the Q268X background as well as the formation of nuclear foci. This was somewhat unexpected because we had previously showed that Q268X appeared to be sequestered in the nuclear membrane and hence not able to interfere with the function of the wt protein (74). Our current results now suggest that there might be physiological conditions in which the MODY1 mutant is localized to a different cellular compartment. It will be of interest in the future to determine whether the changes brought on by PKC signaling contribute in any way to the patients’ diabetes.
The model presented here raises several additional issues for future investigation. The first is, as noted above, the identification of additional factors that affect the final localization of the NRs: the effect of the ‘S78D’ mutation on cytoplasmic localization was greater in RXR and PPAR than in HNF4, suggesting that additional factors may contribute to its nuclear localization. Indeed, NRs are known to contain several NLS and nuclear export signals (20, 58, 75). This became readily evident when we introduced the S78D mutation into the naturally occurring truncation of HNF4 (Q268X). The second is the elucidation of the structural basis for the effect of phosphorylation of S78 on intracellular localization. As noted in the model presented here of the HNF4 DBD, a conformational change may be involved that could impact the ability of the NR to interact with transporter molecules, such as importins. A similar model has been proposed for the effect of phosphorylation on the shuttling of STATs to and from the nucleus (43). The third is the identification of the physiological and pathological signals that trigger PKC phosphorylation as well as determination of the function of the NRs in the cytoplasm—are they nonfunctional aggregates of malfolded proteins or do they have some as-yet-unknown functions? The fourth, and perhaps the most intriguing, is the reason behind the conspicuous absence of a Ser or Thr at position 78 in the classical steroid receptors (Fig. 1). One wonders whether it is related to the fact that localization of these receptors is more dependent upon ligand than it is for nonsteroid receptors. Whatever the outcome of these future studies, it is clear that the conserved Ser between the two zinc fingers is critical to NR function, both genomic and nongenomic, and is affected by the signal transduction pathway.
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MATERIALS AND METHODS |
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-HNF4 [445 (45, 76) and 127 (77)]; rabbit polyclonal -actin (Sigma, St. Louis, MO); mouse monoclonal -mtHSP70 (Affinity BioReagents, Golden, CO); horseradish peroxidase-conjugated goat antirabbit (GR) and goat antimouse (GM) IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); and Alexa Fluor 594 donkey antimouse (DM) and Alexa Fluor 488 GR (Molecular Probes, Eugene, OR). PKC (, ß, and mixture), PKC peptide inhibitor, and PKA/CaMK inhibitor cocktail were from Upstate (Lake Placid, NY). TPA, BisII, phosphatase inhibitor cocktail I and II, cyclohexamide, LMB, and proteasome inhibitor MG132 were from Sigma.
Plasmids
All HNF4 refers to rat HNF41 unless indicated otherwise. Human HNF41 is 100% identical with rat HNF41 in the DBD. The expression vectors containing full-length wt rat HNF41 (GenBank accession no. X57133) [pMT7.HNF41 and pRSET.HNF41 (His.HNF4)] and GST.HNF4.DBD have been described (78, 79) as has the MODY1 mutant Q268X (pMT7.HNF41.Q268X) (74). GST fusions of hRAR.DBD (aa 82–167), hRXR.DBD (aa 130–223), and hTRß.DBD (aa 97–207) have been described (80). N-terminal enhanced GFP (EGFP) fusion proteins were constructed as follows: The open reading frame of HNF41 was amplified from pMT7.HNF41 by PCR using Pfu DNA polymerase and ligated into the pEGFP.C3 vector (Invitrogen, Carlsbad, CA). A full-length human RXR (NM_002957) GFP fusion protein was constructed in a similar fashion by ligating an EcoRI fragment from pMT2.hRXR (45) into pEGFP.C3. A C-terminally truncated human RXR GFP fusion (aa 1–235, RXR.C) was made using an EcoRI-BamHI fragment from the same parental vector. Mouse PPAR (NM_011144) GFP fusion protein was constructed by ligating a BamHI fragment from pSG5.mPPAR (81) into pEGFP.C1 (Invitrogen). Site-directed mutagenesis of all constructs was carried out using the QuikChange kit according to the manufacturer’s protocol (Stratagene, La Jolla, CA) and confirmed by dideoxy sequence (University of California, Riverside, Core Instrumentation Facility). (Sequences of all PCR and mutagenesis primers are given in supplemental Table S1.)
In Vitro Kinase Assay
His-tagged HNF41 (pRSET.HNF41) wt or mutants and GST fusion proteins were expressed in Escherichia coli BL21 (Stratagene) and purified with Talon metal affinity resin (Clontech, Palo Alto, CA) or GSH agarose beads (Sigma), respectively. Phosphorylation by PKC in vitro was carried out in 50-µl reactions as per the manufacturer’s protocol. The phosphorylated proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (Immobilon-P; Millipore, Bedford, MA), stained with Coomassie blue to verify protein loading, and visualized by autoradiography.
Cell Culture, TPA Treatment, Protein Stability Assays, and Immunoblot Analysis
The human hepatoblastoma/hepatocellular carcinoma HepG2 cell line (ATCC HB-8065; American Type Culture Collection, Manassas, VA) and COS-7 cells (ATCC CRL-1651) were maintained at 37 C in 5% CO2 in DMEM containing 10% fetal bovine serum (HepG2) or 10% bovine calf serum (COS-7) and penicillin (100 U/ml) and streptomycin (100 µg/ml). For in vivo stability studies, 2.5 x 105 HepG2 cells were seeded on six-well plates. Sixteen hours later, the cells were treated with 150 nM TPA, 20 µM BisII, and/or 50 µg/ml cyclohexamide as indicated; 0.01% dimethylsulfoxide was the vehicle control. At the indicated time points, the cells were harvested and lysed in RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.05% sodium dodecyl sulfate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride] plus phosphatase inhibitors. Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). Fifteen micrograms of protein from the cleared lysates were subjected to 10% SDS-PAGE, transferred to Immobilon and analyzed by immunoblot (IB) analysis using the color reaction as previously described (79) or enhanced chemiluminescence (ECL) (Amersham Biosciences, Piscataway, NJ). The ECL assay was performed using a 4-h incubation of a 1:5,000 to 1:10,000 dilution of 445 (for HNF4) and a 1:10,000 dilution of GR-horseradish peroxidase. Stability in COS-7 cells was examined in a similar fashion except that 3 x 106 cells were transfected with wt or mutant pMT7.HNF41 (5 µg) in 100-mm plates by calcium phosphate precipitation (45). After 24 h, the cells were trypsinized and replated into 12 wells in six-well plates. Twelve hours later, the cells were treated with 50 µg/ml cyclohexamide (0.1% ethanol was the vehicle control) followed by harvesting in RIPA at the indicated time points and IB analysis as described above.
EMSA
Nuclear extracts from transfected COS-7 cells and TPA-treated HepG2 cells (as above, except cyclohexamide was not added) were analyzed in EMSA as previously described (45). A standard mobility shift reaction (15 µl) contained 1 µg nuclear protein, 1 ng 32P 5'-labeled probe, a double-stranded oligonucleotide corresponding to –85 to –47 of the human apolipoprotein B (ApoB) promoter (79). Dried gels were subjected to autoradiography.
Luciferase Assays
COS-7 cells were seeded onto six-well plates and transfected 24 h later using Lipofectamine 2000 (Invitrogen). Each well received 2 µg luciferase reporter ApoB.-85–47.E4.Luc, 200 ng CMV.ß-gal, and 200 ng wt or mutant pMT7.HNF41 as indicated. ApoB.-85-47.E4.Luc contains the HNF4 response element from the human ApoB promoter (79). The total amount of DNA was normalized by adding empty vector pMT7. Thirty-six hours after transfection, the cells were harvested and lysed in reporter lysis buffer for luciferase measurements (Promega, Madison, WI) and ß-galactosidase activity as described previously (45). Experiments were performed three independent times in duplicate.
Protein Modeling
Three-dimensional (3D) models of the DBD from Rattus rattus HNF4 (rrHNF4) were built by comparative protein structure modeling using the program MODELLER 8v0 (82, 83). The input consisted of the template structure and the alignment of the target sequence with this structure. The output was a 3D model of the target including all nonhydrogen atoms. This model was derived by minimizing violations of distance and dihedral angle restraints extracted from the template structure. The template structure was the RXR from the RXR:RAR DBD heterodimer in complex with the retinoic acid response element DR1 (Protein Data Bank, 1dsz). The rrHNF4 DBD sequence shares 57.1% aa identity to the RXR DBD sequence and is 100% identical with the human HNF4 DBD sequence. Figures were prepared with PyMol (84).
Immunofluorescence Microscopy
COS-7 were seeded on coverslips in 60-mm plates, transfected with the indicated plasmid DNA at nonsaturating conditions (100 ng expression vector per 60-mm plate) using Lipofectamine 2000. Cells were treated with the proteasome inhibitor MG132 (50 µM) for 12 h after the addition of DNA; 12 h later, the cells were washed three times with PBS, fixed in PBS containing 3% formaldehyde for 10 min, washed twice with PBS, permeabilized in cold PBS containing 0.2% Triton X-100 for 5 min, and incubated with PBS containing 5% nonfat milk for 30 min before incubation with the primary antibody as indicated (445, 1:1000; 127, 1:500; -mtHSP70, 1:200) in 1% nonfat milk in PBS overnight. The cells were washed three times with PBS and incubated for 1 h with an appropriate fluorochrome-conjugated secondary antibody (Alexa Fluor 488 GR, 1:500; Alexa Fluor 594 DM, 1:250). Coverslips with stained cells were then washed with PBS and mounted onto glass slides with Krystalon mounting medium (EMD, Gibbstown, NJ) and examined with a Zeiss (Oberkochen, Germany) 510 confocal microscope (x40 or x100 objective) fitted with appropriate fluorescence filters. All localization experiments were performed at least five to 10 independent times and 
100 cells were examined in each experiment (500 to 1000 cells total per construct). All images shown represent 50% of the cells analyzed. Quantification of wt and mutant HNF4 constructs was done using Adobe Photoshop (RGB information, green signal). Measurements from three different parts of each nucleus and cytoplasm were averaged for each of at least 15 randomly chosen cells per construct. All images were corrected for background, using a region of the coverslip containing no cells. Data are expressed as a ratio of cytoplasmic (Fc) to nuclear (Fn) intensity ± SD. The level of expression of the wt and S78D HNF4 constructs in Cos-7 cells under these transfection conditions were comparable with the level of expression of endogenous HNF4 in HepG2 cells and mouse liver as determined by immunoblotting (see supplemental Fig. S6).
For Fig. 5D, HepG2 cells (1 x 106) were plated in 100-mm plates and cultured in DMEM plus 10% fetal bovine serum. Twenty-four hours later, the cells were transfected with 5 µg GFP.HNF4 by Lipofectamine 2000. Twenty-four hours after transfection, the cells (3 x 105) were split into six-well plates with coverslips (12 mm round) precoated with polyethyleneimine (1 mg/ml; Sigma). Twelve hours later, the cells were treated with or without 150 nM TPA as described above (no MG132 was added). At the indicated time points, the coverslips were mounted into a recording chamber holding normal external solution [140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4)]. Visualization of EGFP fluorescence was accomplished using an inverted microscope (TE 300; Nikon, Melville, NY) equipped with wide-field epifluorescence and a standard fluorescein isothiocyanate filter set (Chroma Technology, Rockingham, VT). Xenon arc lamp (100 W) was used as a light source. Images were captured through a x60 plan-achromatic oil immersion objective (1.4 numerical aperture; Nikon). All images were corrected for background, using the fluorescence of a region on the coverslip containing no cells. Data are expressed as a ratio of cytoplasmic (Fc) over nuclear (Fn) EGFP fluorescent emission intensities that were measured and averaged within cellular regions of interest sizing at least 40 x 40 pixels (±SEM). Randomly chosen cells (18–24 per condition) were analyzed for each group; a two-way ANOVA followed by Fisher’s least significant difference test was used to determine significance. All experiments were done at room temperature.
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ACKNOWLEDGMENTS |
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cDNA, S. Green for pSG5.mPPAR, and T. Perlmann for the RXR, RAR, and TRß GST.DBD constructs; L. Shank for preliminary experiments with CRT–/– mouse embryonic fibroblasts; and D. Carter for technical help with confocal microscopy. |
FOOTNOTES |
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Present address for K.S.: Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037.
Disclosure Statement: The authors have nothing to disclose.
First Published Online March 27, 2007
Abbreviations: aa, Amino acid; AF-1, activation function 1; ApoB, apolipoprotein B; AR, androgen receptor; BisII, bisindolylmaleimide II; CRT, calreticulin; 3D, three-dimensional; DBD, DNA binding domain; ECL, enhanced chemiluminescence; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; GFP, green fluorescent protein; GR, glucocorticoid receptor; GST, glutathione S-transferase; HNF4, hepatocyte nuclear factor 4; IB, immunoblot; LBD, ligand binding domain; LMB, leptomycin B; MODY1, maturity onset diabetes of the young 1; mtHSP70, mitochondrial heat shock protein 70; NLS, nuclear localization signal; NR, nuclear receptor; PKA, protein kinase A; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; SXR, steroid X receptor; TPA, phorbol 12-myristate 13-acetate; TR, thyroid hormone receptor; VDR, vitamin D receptor; wt, wild type.
Received for publication July 24, 2006. Accepted for publication March 22, 2007.
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