Article

• The EMBO Journal (2000) 19, 691 - 701
• doi:10.1093/emboj/19.4.691

Activation of orphan receptor-mediated transcription by Ca²⁺/calmodulin-dependent protein kinase IV

Christopher D. Kane¹ and Anthony R. Means¹

1. Department of Pharmacology and Cancer Biology, Duke University Medical Center, PO Box 3813, Durham, NC 27710, USA

Correspondence to:

Anthony R. Means, E-mail: means001@mc.duke.edu

Received 30 July 1999; Accepted 7 December 1999; Revised 17 November 1999

• Keywords:

  • Ca²⁺,
  • CaM-dependent protein kinase IV,
  • calcium,
  • orphan receptor,
  • LXXLL,
  • ROR

Introduction

Elevation of intracellular Ca²⁺ plays a prominent role in a broad array of cellular functions including the activation of transcription and regulation of cellular processes such as the cell cycle and apoptosis (Means, 1995). The Ca²⁺ signal is mediated principally by calmodulin (CaM), which serves as the cell's primary Ca²⁺ receptor in both the cytoplasm and nucleus. Ca²⁺/CaM activates many enzymes including protein kinases and phosphatases, adenylyl cyclases and cyclic nucleotide phosphodiesterases (Means, 1995). Within this group of signaling molecules, significant emphasis has been placed upon the CaM-dependent protein kinases (CaMKs). Three CaMKs, I, II and IV (CaMKI, CaMKII and CaMKIV, respectively), are multifunctional protein Ser/Thr kinases, each of which phosphorylate a variety of substrates in vitro and in vivo (for a review, see Anderson and Kane, 1998). While CaMKI and CaMKII are widely expressed throughout the body, CaMKIV expression is relatively limited to the brain, T-lymphocytes and post-meiotic male germ cells (Ohmstede et al., 1989; Frangakis et al., 1991; Means et al., 1991). CaMKIV is localized predominantly to the nucleus and becomes activated very rapidly upon the elevation of intracellular Ca²⁺ (Jensen et al., 1991). Activation of CaMKIV requires: (i) binding of Ca²⁺/CaM; (ii) phosphorylation of Thr200 in the 'activation loop' by an upstream activating kinase (CaMKK) that is also Ca²⁺/CaM-dependent; and (iii) autophosphorylation of serine residues present in the extreme N-terminus required for relief of a novel form of autoinhibition (Chatila et al., 1996). Truncation of the C-terminal CaM-binding and autoinhibitory domains of CaMKIV results in a Ca²⁺/CaM-independent enzyme whose activity still requires phosphorylation of Thr200 by a CaMKK (Chatila et al., 1996). CaMKIV is a potent activator of several transcription factors including CREB, ATF-1 and SRF (Miranti et al., 1995; Tokumitsu et al., 1995; Sun et al., 1996). CaMKIV-dependent activation of CREB-mediated transcription can occur through its ability to phosphorylate CREB on Ser133 (Matthews et al., 1994). However, CaMKIV may also play another role in CREB-mediated transcription through the activation of the CREB-binding protein (CBP) (Chawla et al., 1998), although the specific mechanism remains to be elucidated.

Our laboratory has evaluated potential roles of CaMKIV through the production of transgenic mice that express a kinase-inactive form of CaMKIV in T-lymphocytes (Anderson et al., 1997) as well as by elimination of the CaMKIV gene locus via homologous recombination (A.R.Means, unpublished data). Analysis of these animals revealed striking phenotypic consequences in neurological, lymphoid and reproductive tissues, correlating with the expression patterns of CaMKIV. Interestingly, the phenotypic consequences of the CaMKIV-deficient animals were similar to those of the staggerer mouse. The staggerer mouse represents a spontaneously arising mutation that has been studied as an animal model of ataxia (Sidman et al., 1962). In addition to the neurological defects, these animals also display immunological and reproductive deficiencies (Trenkner and Hoffmann, 1986; Guastavino and Larsson, 1992). Hamilton et al. (1996) positionally cloned the staggerer mutation and demonstrated that it prevents translation of the ligand-binding domain (LBD) of the retinoic acid receptor-related orphan receptor (ROR). ROR is a member of the thyroid/steroid superfamily of nuclear receptors and is expressed in the Purkinje cells of the cerebellum as well as in lymphoid and reproductive tissues (Giguère et al., 1994; Hamilton et al., 1996; Steinmayr et al., 1998). ROR binds DNA as a monomer and has been shown to drive transcription constitutively from its cognate response element (Giguère et al., 1994, 1995). As this transactivation occurs in the absence of any known ligand, ROR is classified as an orphan receptor. Recently, two independent laboratory groups have disrupted the ROR gene and recapitulated the staggerer phenotype in both sets of mice, demonstrating that functional disruption of the ROR gene is the bona fide target of the staggerer mutation (Dussault et al., 1998; Steinmayr et al., 1998).

Given the ability of CaMKIV to activate transcription factors such as CREB, the overlapping tissue expression patterns of CaMKIV and ROR and the similar phenotypes of the CaMKIV- and ROR-deficient animal models, we tested the ability of CaMKIV to potentiate the transcriptional activity of ROR. We report here that ROR is a Ca²⁺-responsive transcription factor and that ROR, as well as several other orphan receptors, can be activated potently by CaMKIV. Transactivation of ROR is dependent upon its LBD, although the receptor itself is not the target of the kinase. We have identified a class of peptides containing an LXXLL motif that specifically interacts with the LBD of ROR in a CaMKIV-dependent manner. These peptides can serve as potent antagonists of ROR- and other orphan receptor-mediated transcription in both the absence and presence of CaMKIV. Our findings suggest a novel paradigm governing the transactivation of a select group of orphan receptors, in which biological stimuli that mobilize Ca²⁺ signaling cascades can markedly increase orphan receptor-mediated transcription via the activation of CaMKIV. Such a mechanism provides an alternative means by which these orphan receptors, when co-expressed in cells with CaMKIV, may drive transcription in the apparent absence of any cognate ligands.

ROR is activated by CaMKIV and is a Ca²⁺-responsive transcription factor

Based upon the similarity of phenotypes seen in staggerer and CaMKIV-deficient animals, we asked whether CaMKIV could increase transcription mediated by ROR. Thus, we transiently transfected the human keratinocyte cell line HaCaT with an expression vector for human ROR and its cognate luciferase-based reporter containing three copies of a consensus ROR response element in front of the thymidine kinase minimal promoter [RORE(3)TKluc]. As reported previously for this receptor, ROR displays constitutive and ligand-independent transcriptional activity (Figure 1A, open bar) (Giguère et al., 1994).

Figure 1.

Transcriptional activation of ROR by CaMKIV. (A) HaCaT cells were transfected with 4 g of RORE(3)TKluc reporter and 1 g of ROR expression vector in either the absence or presence of 4 g of the Ca²⁺/CaM-independent (CaMKIV 1–317) or catalytically inactive (K75E CaMKIV 1–317) CaMKIV expression vectors. Cells were harvested after 36 h in culture and luciferase activity is reported as corrected relative light units. (B) Ca²⁺ influx mediated by ionomycin treatment induces ROR transcriptional activity. HaCaT cells were transfected with reporter and ROR expression vector and after 16 h in culture were treated with 0.67 M ionomcyin, 30 M KN-93 or both ionomycin and KN-93. Cells were stimulated for an additional 24 h, harvested and the luciferase activity reported as corrected relative light units.

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To determine if CaMKIV could potentiate this activity further, an expression vector encoding Ca²⁺/CaM-independent CaMKIV (CaMKIV 1–317) was co-transfected with the ROR expression vector. Quite surprisingly, CaMKIV activated transcription from RORE(3)TKluc an additional 20- to 30-fold over the activity of ROR alone (Figure 1A, black bar). Co-transfection of a catalytically inactive expression vector of CaMKIV (K75E CaMKIV 1–317) failed to produce any additional activation above that of ROR's constitutive activity, indicating that CaMKIV protein kinase activity was required for ROR activation (Figure 1A, striped bar) while co-transfection of CaMKIV 1–317 modestly activated reporter alone (Figure 1A, speckled bar). Because the ROR expression vector used in these experiments was driven by the cytomegalovirus (CMV) promoter, and because this promoter has recently been shown to be Ca²⁺ sensitive, we produced constructs derived from the Ca²⁺-insensitive Rous sarcoma virus (RSV) and SV40 promoters and compared them with CMV-driven ROR in the absence and presence of CaMKIV 1–317. Each ROR expression vector displayed statistically indistinguishable constitutive activity and each was significantly induced by CaMKIV 1–317 (data not shown), suggesting that the activation of transcription is not due to an indirect effect of the kinase on the ROR expression vector. Finally, CaMKIV-dependent activation of ROR was also found to occur in other cell lines including the human T-cell line Jurkat and the rat pituitary cell line GH3 (data not shown). In each instance, marked activation of ROR occurred in the presence of Ca²⁺/CaM-independent CaMKIV, indicating that this effect was not cell line specific.

While it was clear that ROR could be activated by Ca²⁺/CaM-independent CaMKIV, we wanted to determine if ROR activity could be modulated by Ca²⁺ signaling. Therefore, HaCaT cells were transfected with ROR and subsequently were stimulated with the Ca²⁺-elevating agent ionomycin. Ionomycin treatment had a negligible effect upon reporter alone (Figure 1B, left hand side). However, treatment with ionomycin potently activated ROR. This activation resulted in nearly a 12-fold activation of ROR in the presence of Ca²⁺ versus its activity in the presence of dimethylsulfoxide (DMSO) (Figure 1B, right hand side, open versus stippled bar).

HaCaT cells express endogenous CaMKIV based upon their expression of CaMKIV mRNA as detected via RT–PCR and Northern analysis (C.D.Kane and A.R.Means, unpublished data). In addition to CaMKIV, it is possible that the ubiquitously expressed CaM kinases I and II as well as other CaM-dependent enzymes are also expressed in these cells. In order to identify a role for a CaM kinase in ROR activation, KN-93, a selective inhibitor of CaMKI, II and IV, was added concomitantly with iono-mycin to cells transfected with ROR. While KN-93 did not adversely effect the constitutive activity of ROR (Figure 1B, black bar), the drug potently repressed the ability of ionomycin to activate ROR (Figure 1B, striped bar). These results show that a CaM kinase inhibitor can block Ca²⁺-dependent activation of ROR, suggesting that activation of ROR-mediated transcription by Ca²⁺ is occurring via activation of a CaM kinase.

CaMKIV and I, but not II, can activate ROR

In order to determine which members of the CaM kinase family could activate ROR transcription, HaCaT cells were transiently transfected with expression vectors encoding Ca²⁺/CaM-independent CaM kinase I, II or IV. As previously seen in Figure 1, CaMKIV potently activated ROR-mediated transcription 20- to 30-fold more than that seen by ROR alone (Figure 2, black versus open bar). Activation was also obtained with Ca²⁺/CaM-independent CaMKI (Figure 2, shaded bar). The different efficacies of ROR activation in response to CaMKIV and CaMKI may reflect differences in the relative expression levels of the transfected kinase cDNA constructs in HaCaT cells. In contrast, Ca²⁺/CaM-independent CaMKII failed to demonstrate any activation and actually seemed to repress ROR's constitutive activity (Figure 2, wavy bar). Indeed, in contrast to CaMKIV and CaMKI, CaMKII has been shown potently to repress transcriptional activation by several transcription factors (Matthews et al., 1994; Nghiem et al., 1994; S.S.Hook, C.D.Kane and A.R.Means, unpublished observations). Catalytically inactive mutants of CaMKIV and CaMKII (K75E CaMKIV 1–317 and K42M CaMKII 1–290) failed to activate ROR (Figure 2, lateral and vertical striped bars, respectively). These results demonstrate a clear CaM kinase specificity for ROR activation, with CaMKIV and CaMKI acting to stimulate ROR-mediated transcription while CaMKII fails to do so.

Figure 2.

Calmodulin kinase specificity for activation of ROR-mediated transcription. HaCaT cells were transfected with reporter and 1 g of ROR expression vector in either the absence or presence of 4 g of each CaM kinase expression vector. The kinases included Ca²⁺/CaM-independent CaMKIV (CaMKIV 1–317), catalytically inactive CaMKIV (K75E CaMKIV 1–317), Ca²⁺/CaM-independent CaMKI (CaMKI 1–294), Ca²⁺/CaM-independent CaMKII (CaMKII 1–290) and catalytically inactive CaMKII (K42M CaMKII 1–290). Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as corrected relative light units.

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Activation of transcription by CaMKIV is selective for orphan nuclear receptors

Next we examined if activation of ROR-mediated transcription was specific for ROR or was a property shared by several classes of nuclear receptors. We therefore surveyed a cohort of nuclear receptors for their ability to be activated by CaMKIV. Several different orphan receptors were transiently transfected into HaCaT cells in the absence or presence of CaMKIV 1–317. The RORE(3)TKluc reporter was used for each of these receptors as they have all been shown previously to support transcription from this reporter (Giguère et al., 1994; Schräder et al., 1996). In addition to ROR1, the receptors tested included an N-terminal splice variant of ROR1 termed ROR2, a separate ROR gene family member termed ROR and an unrelated orphan receptor COUP-TFI. As seen in Figure 3A (left hand side), all of these receptors display some ligand-independent, constitutive activity, as has been reported previously (Giguère et al., 1994; Medvedev et al., 1996; Schräder et al., 1996). Co-transfection of CaMKIV 1–317 resulted in marked activation of all the orphan receptors tested (Figure 3A, right hand side). The fold activation for the ROR orphan receptors was generally in the range of 20- to 40-fold induction, while that for COUP-TF1 was in the 7- to 10-fold range.

Figure 3.

Orphan receptor specificity for transcriptional activation by CaMKIV. (A) HaCaT cells were transfected with 4 g of RORE(3)TKluc reporter and 1 g of the appropriate orphan receptor expression vector in both the absence and presence of 4 g of CaMKIV 1–317 expression vector. Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as corrected relative light units. (B) Jurkat cells were transfected with 12 g of TREpal(2)luc reporter and 4 g of TR expression vector in the absence or presence of 4 g of either CaMKIV 1–317 or K75E CaMKIV 1–317. Cells were treated with 150 nM T3 for 36 h, harvested and the luciferase activity reported as corrected relative light units. (C) HepG2 cells were transfected with 1.5 g of ERETKluc and 1 g of ER expression vector in the presence of 4 g of either CaMKIV 1–317 or K75E CaMKIV 1–317. Cells were treated with 100 nM 17-estradiol for 24 h, harvested and the luciferase activity reported as corrected relative light units.

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Next we tested the ability of CaMKIV to potentiate the activity of two prototypical nuclear receptors, the thyroid hormone receptor (TR) and the estrogen receptor (ER). Transient transfection of Jurkat cells with an expression vector for TR and treatment with 150 nM T3 resulted in a large T3-dependent induction of TR-mediated transcription (Figure 3B, open bars). If CaMKIV 1–317 was co-transfected with TR, no potentiation of TR-mediated transcription occurred in either the absence or the presence of T3 (Figure 3B, black bars). Likewise, co-transfection with K75E CaMKIV 1–317 also had no significant effects upon TR-mediated transcription (Figure 3B, striped bars)

Similar experiments were performed using ER to test for transcriptional potentiation in the presence of CaMKIV. As seen in Figure 3C (left side), the presence of CaMKIV 1–317 did not potentiate the ligand-independent activity of ER. CaMKIV also failed to provide any additional potentiation of the 17-estradiol-dependent activity of ER (Figure 3C, right side). These findings demonstrate that the presence of CaMKIV does not change either the ligand-dependent or ligand-independent transactivation of the thyroid or estrogen nuclear receptors and that the ability of CaMKIV to activate transcription seems to be limited to a distinct group of orphan receptors.

Phosphorylation of the N-terminus of ROR is not required for CaMKIV-dependent transactivation

Previous studies by Giguère et al. (1994, 1995; McBroom et al., 1995) demonstrated a regulatory region within the ROR N-terminal domain that influences the specificity and efficiency of binding to DNA. In addition, this domain contains several putative phosphorylation sites for multiple protein kinases. Two sites, Ser49 and Ser58, are found within this regulatory domain and match the consensus recognition motifs for CaMKIV (Giguère et al., 1995; White et al., 1998). In order to test if these residues are required for CaMKIV-dependent activation of ROR, a double point mutant, S49E/S58E ROR (S49/58E ROR), was used in the transient transfection assay. As shown in Figure 4 (left hand side), both the wild-type and S49/58E ROR have identical constitutive activities on the reporter. Co-transfection of CaMKIV 1–317 resulted in potent activation of both receptors (Figure 4, right hand side). The overall transcriptional activity of S49/58E ROR was slightly greater than that of the wild-type receptor in the CaMKIV-activated state. However, the overall fold induction of both wild-type and S49/58E ROR by CaMKIV was similar (23-fold versus 30-fold, respectively). This observation was corroborated by an additional experiment in which an N-terminal truncation mutant (23–74 ROR) also displayed robust activation after co-transfection with CaMKIV (data not shown). These experiments suggest that the putative CaMKIV phosphorylation sites found within the N-terminal domain of ROR are not required for the CaMKIV-dependent activation of ROR-mediated transcription.

Figure 4.

Transcriptional activation of wild-type or S49/58E ROR by CaMKIV. HaCaT cells were transfected with reporter and 1 g of wild-type or S49/58 ROR in the absence or presence of 4 g of CaMKIV 1–317. Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as corrected relative light units.

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Enhancement of ROR-dependent transactivation by CaMKIV is mediated through the LBD

While there is significant variability in the primary amino acid sequences of the LBDs of the various members of the nuclear/steroid receptor superfamily, crystallographic analysis of these domains from PR, ER, PPAR, RXR and TR have shown that the principal folding features are conserved (Wurtz et al., 1996). Structural and mutagenesis studies of liganded receptors have suggested a key role for the activation function 2 (AF-2) domain, a conserved region within the LBD structure corresponding to helix 12 that forms an interface with members of the coactivator class of nuclear receptor-interacting proteins such as SRC1, GRIP1 and pCIP (Oñate et al., 1995; Hong et al., 1996; Wurtz et al., 1996; Torchia et al., 1997). Deletion analysis of ROR demonstrates that this receptor has a functional AF-2 domain within its LBD. Removal of this domain renders ROR transcriptionally inactive and results in the staggerer phenotype (McBroom et al., 1995; Hamilton et al., 1996; Harding et al., 1997). In order to address if the LBD of ROR is sufficient to mediate CaMKIV-dependent activation of transcription, the LBD of ROR was fused in-frame to the DNA-binding domain (DBD) of the yeast transcription factor Gal4 (Gal4:RORLBD). This construct or Gal4DBD alone was co-transfected into GH3 cells with a 5 Gal4UAS/TATA/luciferase reporter in the absence or presence of CaMKIV 1–317. As seen in Figure 5, Gal4:RORLBD exhibits constitutive transcriptional activity and can be activated upon co-transfection with CaMKIV, while the Gal4DBD is transcriptionally silent under all conditions tested. These results demonstrate that the effects of CaMKIV on full-length ROR can be recapitulated with an RORLBD chimera. Thus, the LBD of ROR is sufficient for mediating the CaMKIV-dependent activation of transcription.

Figure 5.

Transcriptional activation of a Gal4:RORLBD chimera. GH3 cells were transfected with 3 g of 5 Gal4UAS/TATA/luciferase reporter and 2 g of Gal4DBD or Gal4:RORLBD in the absence or presence of 4 g of CaMKIV 1–317. Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as corrected relative light units.

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The ligand-binding domain of ROR is not phosphorylated

As the LBD of ROR is capable of being potentiated by co-transfection with CaMKIV, we wanted to determine if the LBD of ROR is a direct target for phosphorylation by CaMKIV. Therefore, the entire LBD of ROR (amino acids 270–524) was expressed as a GST fusion protein in Escherichia coli (GST:RORLBD), purified to homogeneity and used as a substrate for an in vitro kinase assay. Given the overlapping substrate specificity of CaMKI and IV, as well as the ability of each of these kinases to activate ROR in transient transfections and the fact that the kinetics of peptide substrate phosphorylation are greater for CaMKI than they are for CaMKIV, CaMKI was chosen for the in vitro phosphorylation reaction.

As shown by the autoradiogram in Figure 6A (upper panel), GST:RORLBD was not phosphorylated in the presence of either kinase reaction buffer or CaMKI. CaMKI can be activated further by the addition of CaMKKB, which phosphorylates Thr177 of CaMKI. This activation loop phosphorylation significantly enhances the kinetics of CaMKI substrate phosphorylation and increases its specific activity 25-fold (Haribabu et al., 1995). The addition of CaMKKB to CaMKI resulted in the phosphorylation of CaMKI, yet CaMKI failed to recognize GST:RORLBD as a substrate (Figure 6A, upper panel). Likewise, CaMKKB alone also failed to phosphorylate GST:RORLBD. Both enzymes were shown to be active, as control reactions run in parallel with a peptide corresponding to the N-terminal 1–117 amino acids of P300 (1–117:P300) demonstrated that CaMKI was active on its own and that addition of CaMKKB resulted in a marked increase in CaMKI activity (Figure 6B). Coomassie staining of the fractionated kinase reactions indicated that GST:RORLBD was present in equal amounts for all reaction conditions (Figure 6A, bottom panel). These results demonstrate that the LBD of ROR is not an in vitro substrate for CaMKI and suggest that the mechanism of CaM kinase activation of ROR is not via direct phosphorylation of ROR itself. This is supported by the failure of either CaMKI or CaMKIV to phosphorylate in vitro transcribed and translated full-length ROR (data not shown).

Figure 6.

In vitro phosphorylation of GST:RORLBD. (A) The ligand-binding domain of ROR was used as a substrate for in vitro kinase assay using recombinant CaMKI and KKB. All recombinant proteins were expressed and purified as fusion proteins with either GST (RORLBD and CaMKI) or maltose-binding protein (KKB). Upper panel: an autoradiogram of kinase reactions after fractionation on a 12% SDS–polyacrylamide gel. GST:RORLBD (500 ng) was incubated in the absence (-) or presence (+) of CaMKI (200 ng) or KKB (500 ng) in kinase reaction buffer for 20 min. The positions of CaMKI and GST:RORLBD are denoted by arrows. CaMKI does not undergo autophosphorylation but will become phosphorylated in the presence of KKB. Lower panel: Coomassie staining of the gel used for autoradiography. Arrows to the right denote the position of CaMKI and GST:RORLBD. (B) His-tagged 1–117:P300 serves as a positive control for CaMKI activity. Kinase assays were performed as described in (A) using 1–117:P300 (500 ng) as a substrate. The panel displays an autoradiogram of kinase reactions after fractionation on SDS–PAGE. Coomassie staining indicated that the peptide was present in equal amounts for all reactions (data not shown).

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Detection of RORLBD-interacting peptides

The nuclear receptor coactivators have been found to contain a signature LXXLL motif, also referred to as an 'NR-box' (Heery et al., 1997; Torchia et al., 1997). In an attempt to identify LXXLL-containing peptides that specifically interact with liganded ER, Chang et al. (1999) used a combinatorial phage display approach and discovered three classes (class I, II and III) of small, 19 amino acid peptides that specifically bound ER in an agonist-dependent manner. Representative members of each of these classes as well as LXXLL-containing peptides from the coactivators P300, GRIP1 and SRC1 were tested in the context of the mammalian two-hybrid assay in order to identify peptides that could interact with the LBD of ROR in either the absence or presence of CaMKIV. A series of peptides containing a central LXXLL motif were expressed as fusion proteins with the Gal4DBD. The entire ROR LBD was expressed as a fusion protein with the VP16 activation domain (VP16:RORLBD) and these two plasmids were transiently transfected into HaCaT cells in the absence or presence of CaMKIV 1–317. A single class of VP16:RORLBD-interacting peptides was identified. As seen in Figure 7A (left hand side), the peptides fused to Gal4DBD did not support basal transcription by themselves. When co-transfected in the presence of VP16:RORLBD, two members of the class II peptides showed weak interaction (Figure 7A, D47 and C33, middle section). Surprisingly, co-transfection of CaMKIV 1–317 resulted in a robust increase in the interaction between class II peptides D47/C33 and VP16:RORLBD that was not seen with the non-interacting class III peptide F6 (Figure 7A, right hand side). The class II peptides D47 and C33 were the only fusion proteins that displayed basal and CaMKIV-dependent interaction. Other peptides containing LXXLL motifs from P300, GRIP-1 and SRC1 failed to interact with the RORLBD in either the absence or presence of CaMKIV (data not shown). These results suggest that the LBD of ROR is necessary and sufficient for interaction with peptides containing the LXXLL nuclear receptor-interacting motif and that CaMKIV can increase these interactions in the context of a mammalian cell.

Figure 7.

Mammalian two-hybrid assay using LXXLL-containing peptides and CaMKIV 1–317. (A) HaCaT cells were transfected with 3 g of 5 Gal4UAS/TATA/luc with 2 g of Gal4DBD fusion of the LXXLL-containing peptides D47, C33 and F6 either alone or in the presence of 2 g of VP16:RORLBD or with both VP16:RORLBD and 4 g of CaMKIV 1–317. Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as corrected relative light units. (B) Transfections were performed as in (A) using 2 g of VP16:ERHBD. Cells were maintained in phenol red-free MEM with 10% charcoal-stripped FBS at all times. Approximately 16 h after transfection, cells were treated with 100 nM -estradiol or ethanol for 24 h, harvested and the luciferase activity reported as corrected relative light units.

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In order to strengthen the correlation between CaMKIV activity and the modulation of orphan receptor activity, we assessed the peptide-binding activities of ER in the context of the mammalian two-hybrid assay in the absence or presence of CaMKIV. Recalling that ER is unresponsive to CaMKIV (Figure 3C), we obtained an expression vector containing the VP16 activation domain fused to the entire hormone-binding domain (HBD) of ER (VP16:ERHBD). Chang et al. (1999) have shown that the peptide-binding specificity of ER is the converse of what is observed for ROR, i.e. -estradiol-bound VP16:ERHBD binds weakly to D47 and strongly to F6. This result is recapitulated in Figure 7B (left hand side) in which agonist-bound VP16:ERHBD interacts weakly with D47 and strongly with F6, whereas no interactions occur in the absence of ligand. Co-transfection with CaMKIV 1–317 fails to increase the interactions between VP16:ERHBD and D47, while causing a modest (<1-fold) increase of interaction for F6 (Figure 7B, right hand side). The failure of CaMKIV to cause a significant increase in the interaction between the peptides and the HBD of ER is consistent with the selectivity seen for the activation of distinct classes of nuclear receptors by CaMKIV.

Class II LXXLL-containing peptides can disrupt ROR-mediated transcription

It has been suggested that LXXLL-containing peptides interact with the AF-2 region of nuclear receptors by mimicking interactions used by full-length coactivators (Heery et al., 1997; Torchia et al., 1997; Nolte et al., 1998; Chang et al., 1999). Based on these data, we thought it likely that the class II peptides should also interact with the AF-2 region of ROR and that, if so, they should act as dominant-negative inhibitors of ROR-mediated transcription. In order to investigate this hypothesis, ROR was co-transfected with CaMKIV 1–317 in HaCaT cells in the presence of increasing input DNA encoding either the ROR-interacting peptide D47 or the non-interacting peptide F6. Luciferase activity was plotted as the percentage of maximal ROR-dependent activity seen in the presence of CaMKIV. As seen in Figure 8A, D47 was a potent inhibitor of CaMKIV-dependent, ROR-mediated transcription, while the non-interacting peptide F6 had no effect (filled versus open circles, respectively). Similar results were obtained using D47 and F6 in the absence of CaMKIV. Constitutive ROR transcription was suppressed in the presence of D47, while F6 had no effect (data not shown). Repression of ROR-mediated transcription was only observed with the VP16:RORLBD-interacting peptide D47, suggesting that this protein may be preventing the interaction of ROR with a putative endogenous coactivator.

Figure 8.

Certain LXXLL-containing peptides antagonize ROR-mediated transcription. (A) HaCaT cells were transfected with 4 g of RORE(3)TKluc reporter, 1 g of ROR and 4 g of CaMKIV 1–317 in the absence or presence of increasing concentrations of either D47 or F6 competitor DNA. Cells were maintained in culture for 36 h, harvested and the luciferase activity reported as a percentage of the maximal, CaMKIV-dependent luciferase activity that occurs when competitor is absent. (B) Transfections were performed and analyzed as in (A), except using 1 g of ROR or COUP-TF1 in the presence of increasing concentrations of D47 competitor DNA.

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In order to observe if D47-dependent repression of ROR was unique to this receptor or if it was common to other orphan receptors, a similar repression experiment was carried out with the orphan nuclear receptors ROR and COUP-TFI. As seen in Figure 8B, D47 was a potent inhibitor of CaMKIV-dependent ROR-mediated transcription and also repressed COUP-TFI, but to a lesser extent (filled versus open circles). While ROR was repressed to 25% of its maximal activity and to an extent similar to ROR, COUP-TFI was repressed to only 60% of its maximal activity. This may be due to the fact that on the RORE(3)TKluc reporter, ROR and ROR bind as monomers and may be repressed efficiently by a single copy of D47. In contrast, COUP-TFI will bind as either a homodimer or a heterodimer with RXR (Cooney et al., 1992) and, therefore, may not be repressed as efficiently by a single copy of D47. Collectively, these results suggest that the ability of D47 to repress ROR, ROR and COUP-TFI is a common property, and imply that D47 is competing for a specific coactivator that is present in HaCaT cells and required for the transcriptional activity of these orphan receptors.

The term orphan receptor has been developed to describe those nuclear receptors for which there are no known ligands. Unlike the traditional thyroid/steroid class of nuclear receptors that require specific ligands to support transcription, the orphan receptors display constitutive activity that is thought to be ligand independent. Our experiments have indicated that an established signaling pathway can potently activate orphan receptor-mediated transcription in a previously undescribed manner. We have shown that ROR is a Ca²⁺-responsive transcription factor whose constitutive activity is significantly enhanced by Ca²⁺ influx and that this increase of activity requires CaM kinase activity. In this respect, ROR joins CREB as a facilitator of Ca²⁺-induced gene expression. Many biological stimuli including hormones, growth factors and T-cell receptor engagement induce Ca²⁺ signals and subsequent activation of transcription (Means, 1995). In those tissues that co-express ROR and CaMKIV, such stimuli would be predicted to be significant activators of RORE-responsive genes. The identification of RORE-containing genes in tissues expressing CaMKIV will be a prerequisite for testing this hypothesis in vivo.

Covalent modification via phosphorylation is known to be a potent mechanism for activation of transcription factors such as CREB and the CREB family members (Gonzalez and Montminy, 1989; Sun et al., 1996). In stimulated cells, CaMKIV and other CREB kinases are known to phosphorylate CREB on Ser133. This phosphorylation event is required for the recruitment of the coactivator CBP, which nucleates a multiprotein complex that ultimately produces a large increase of CREBmediated transcription (Chrivia et al., 1993). Stimulation of nuclear hormone receptors occurs through the introduction and binding of their cognate ligands. Ligand binding dissociates bound co-repressor molecules, which allows recruitment of coactivator molecules that support transcription. Like CREB, several of the nuclear receptors including PR, ER, ER and PPAR undergo phosphorylation (for a review, see Weigel and Zhang, 1998). The receptors are often multiply phosphorylated on serine and threonine residues by several different kinases including PKA and the MAP kinases (Aronica and Katzenellenbogen, 1993; Bunone et al., 1996; Weigel and Zhang, 1998). In general, nuclear receptor phosphorylation affects DNA and/or ligand binding, resulting in either modest potentiation or repression of transcription (Aronica and Katzenellenbogen, 1993; Hu et al., 1996; Takimoto et al., 1996). Our studies indicate that the mechanism of ROR activation is unlikely to include the direct phosphorylation of ROR by CaMKIV. Mutation of potential CaMKIV phosphorylation sites does not prevent the ability of CaMKIV to activate ROR (Figure 4). Additionally, CaMKI will not phosphorylate the LBD of ROR in vitro (Figure 6A), further suggesting that the target of CaM kinase activity is not ROR itself.

The naturally occurring staggerer mutation and deletion analysis of ROR, as well as our own work (Figure 5), demonstrates that the AF-2 domain of the ROR LBD is required for transcriptional activity. In the context of the mammalian two-hybrid assay performed in HaCaT cells, the LBD of ROR is sufficient for interaction with specific LXXLL-containing peptides. Co-transfection of CaMKIV increases this interaction significantly, demonstrating that the LBD is a sufficient platform for supporting CaMKIV-dependent induction of transcription. Direct phosphorylation of the interacting LXXLL peptides by CaMKIV is not possible, as they do not contain the RXXS substrate recognition motif required for substrates of CaMKIV (Table I). This finding strongly suggests that CaMKIV is phosphorylating and activating another protein(s) that is serving as a bridge or scaffold between the LBD and the interacting class II peptides.

Table 1: RORLBD interaction motifs

View full table

Potential candidates for the 'bridge' between ROR and the class II ROR-interacting peptides are the co-integrator molecules CBP and/or P300, which act as transcriptional adaptors for a plethora of transcription factors (Chrivia et al., 1993; Eckner et al., 1994). The adenoviral oncoprotein E1A can bind to and inhibit CBP/P300 function (Arany et al., 1995). Therefore, cotransfection of an expression vector encoding E1A commonly is used to disrupt CBP/P300-dependent transcription. Indeed, co-transfection of E1A represses >95% of the CaMKIV-dependent activation of ROR, implicating CBP/P300 in ROR-mediated transcription (data not shown). However, in contrast to a recent report which described the interaction between the N-terminus of P300 and ROR in JEG-3 cells (Lau et al., 1999), mammalian two-hybrid analysis using the LXXLL domain of P300 (amino acids 74–92) or larger fragments of P300 (amino acids 1–117 or 1–595) failed to interact with VP16:RORLBD in HaCaT cells (Table I and data not shown). Clearly, in HaCaT cells, CBP/P300 is required for ROR function but associates with ROR indirectly, suggesting a supporting role for this co-integrator in ROR-mediated transcription.

Analysis of the class II peptides that interact with ROR's LBD has allowed the creation of an ROR interaction motif where the primary determinants appear to be HVXXHPLLØXLL (Table I). Note that the coactivators that failed to interact with ROR's LBD in HaCaT cells (P300, SRC-1 and GRIP1) do not contain a proline residue at the -2 position relative to the LXXLL sequence. The ability of the class II peptides to interact with ROR's LBD would imply that a proline at the -2 position is a key determinant of coactivator–orphan receptor binding specificity. Pattern Hit Initiated (PHI)-BLAST searches of the sequence database using this consensus and the peptide sequences of C33 and D47 reveal that there are currently no statistically significant identities between the peptides and the deposited sequences. This raises the exciting possibility that a novel, 'class II-like' coactivator molecule exists for the orphan receptors that has yet to be identified. Such a factor would be expected to be expressed in multiple tissues and to interact stably and constitutively with ROR, based upon the ability of ROR to drive transcription in the absence of CaMKIV in at least three different cell lineages (epidermal, lymphoid and neuronal; data not shown). There is current biological precedence for a nuclear receptor that is constitutively active in the absence of naturally occurring ligands. The orphan receptor CAR- (constitutive androstane receptor-) has been shown to exhibit ligand-independent transcription by virtue of the ability of its LBD to interact stably with the coactivator SRC-1 in the absence of exogenous hormone (Forman et al., 1999). CAR- and ROR may be mechanistically similar with respect to their constitutive interaction with coactivator molecules and transcriptional activity in the absence of naturally occurring ligands.

Further evidence for the existence of a 'class II-like' coactivator is found in the ability of the D47 peptide to serve as an efficacious antagonist of orphan receptor-mediated transcription. Both ROR and ROR, and to a lesser extent COUP-TFI, are repressed by co-transfection of D47. This broad spectrum inhibition would suggest that D47 may be competing with an endogenous coactivator found in HaCaT cells for orphan receptor binding. If so, this orphan receptor-specific coactivator could itself be a potential target for CaMKIV, in addition to that of the putative co-integrator molecule(s) that has been implicated in orphan receptor-mediated transcription as defined previously in the mammalian two-hybrid experiments. In addition, these studies suggest that the class II peptides could be used as peptide antagonists of orphan receptor-mediated transcription. As such, these peptides could be powerful tools in the study of orphan receptor-related biology or as possible therapeutic agents in cases of orphan receptor-mediated disease.

In recent years, the physiological ligands for several former orphan receptors have been discovered and found to be metabolites of lipophilic and/or steroid molecules. These include 9-cis retinoic acid for RXR (Heyman et al., 1992), polyunsaturated lipids and their metabolites for PPARs (Göttlicher et al., 1992), and, most recently, chenodeoxycholic acid and other bile acids for the farnesyl X receptor (Makishima et al., 1999; Parks et al., 1999). It remains probable that several of the current orphan receptors will eventually be shown to require ligands in order to stimulate transcription. In this regard, an alternative explanation concerning the ability of CaMKIV to activate ROR that has not been addressed in our studies is the possibility that activation of CaMKIV results in creation of the physiological ligand of ROR. Such a process could be due to the ability of CaMKIV to covalently modify and therefore regulate the activity of a biosynthetic enzyme involved in either the production or destruction of a currently unidentified, naturally occurring ligand for the orphan receptors examined here. Confirmation of these ideas awaits the time that this currently hypothetical ligand has been isolated and fully characterized as a bona fide ligand of ROR. Based upon this work, we can propose at least two possible models for activation of ROR-mediated transcription by CaMKIV. In the first model, CaMKIV mediates its positive effects on transcription through the covalent modification of coactivator molecules such as the putative 'class II-like' coactivator and/or CBP/P300. The second model of CaMKIV activation could occur at the level of the LBD of ROR in a manner reminiscent of the traditional nuclear receptors. In this scenario, cellular stimulation and CaMKIV activation would modulate the activity of a biosynthetic enzyme which modifies or produces a lipophilic molecule that serves as a physiological ligand of ROR. Ligand binding to ROR would nucleate a complex between the receptor, the 'class II-like coactivator' and a co-integrator molecule such as CBP/P300, as is seen with the thyroid/steroid class of nuclear receptors. It should be noted that these models describing the CaMKIV-dependent activation of ROR and the other orphan receptors are not mutually exclusive and could easily utilize components of each of the postulated mechanisms in order to achieve maximal receptor activation. In an effort to delineate which of these pathways are at work in response to CaMKIV activation, we are currently in the process of identifying and characterizing the molecular mechanism by which CaMKIV mediates orphan receptor-dependent transcription.

Plasmids

Expression vectors in the CMX background containing the cDNAs for ROR (ROR1), 23–74 ROR, ROR2, ROR, as well as the reporter RORE(3)TKluc have been described previously and were kindly provided by V.Giguère (Giguère et al., 1994, 1995; Medvedev et al., 1996). The site-specific mutant S49/58E ROR was generated via the Kunkel method of mutagenesis using a synthetic oligonucleotide containing the mutations of interest and was confirmed by sequencing. RSV-COUP-TF1 (a gift of A.Cooney) and RSV:TR, TREpal(2)luc, RSV:ER and ERE(1)TKluc were provided by D.McDonnell. The Ca²⁺/CaM-independent and catalytically inactive expression vectors of pSG5:CaMKIV 1–317, pSG5:CaMKII 1–290 and SR:CaMKI 1–294 have also been described previously (Sun et al., 1995; Chatila et al., 1996; Anderson et al., 1998). The His-tagged 1–117:P300 plasmid was created via PCR amplification of the N-terminus of human P300 (amino acids 1–117), and the subsequent product was subcloned into the prokaryotic expression vector pet30(c) (Novagen). The plasmid GST:RORLBD was generated by amplifying the LBD (amino acids 270–524) of CMX:ROR with Pfu polymerase and subcloning the product into the GST expression vector pGEXKG. Gal4:RORLBD and VP16:RORLBD were created by subcloning the LBD of GST:RORLBD into the EcoRI–BamHI sites of either the Gal4DBD (pM) plasmid or the VP16 activation domain plasmid of the mammalian two-hybrid system (Clontech). The 19 amino acid LXXLL-containing domain of human P300 (amino acids 74–92) was amplified using similar procedures and subcloned into the SalI–BamHI sites of pM. Plasmids pM:D47, pM:C33, pM:F6, pM:SRC1 (amino acids 629–769), pM:GRIP1 (amino acids 634–760) and VP16:ERHBD were provided by D.McDonnell and have been described previously (Chang et al., 1999). All PCR products were sequenced to verify their nucleotide sequences.

Transfections

HaCaT cells were transiently transfected using the DEAE–dextran method. Cells were plated at a density of 250 000 cells/well (6-well plates) in 90% minimal essential medium (MEM) and 10% fetal bovine serum (FBS) (Gibco-BRL) in 1 penicillin/streptomycin and maintained at 37°C and 5% CO₂ for 18–24 h. DNAs (see figures) and an additional 250 ng of RSV:LacZ (for transfection efficiency) were added to 1 Hank's buffered saline solution (HBSS) and DEAE–dextran (Sigma), mixed and added to twice washed cells (HBSS) overlaid with 100% MEM with 100 M choloroquine for 3 h. Afterwards, cells were glycerol shocked, washed and maintained in 90% MEM/10% FBS for 36 h until harvest. Jurkat cells and GH3 cells were transfected via electroporation (Chatila et al., 1996; Sun et al., 1996) and HepG2 cells via lipofectamine (Norris et al., 1998) as previously described. In those experiments requiring stimulants, cells were allowed to recover from the initial transfection for 12–18 h and were subsequently treated with 0.67 M ionomycin (Calbiochem) or 30 M KN-93 (Calbiochem), or an equal volume of DMSO carrier. Cells were treated for 24 h and then harvested. Transfections were performed in triplicate for each condition tested on at least three different occasions.

Luciferase and CPRG assays

Cells were washed in 1 phosphate-buffered saline (PBS) and lysed on ice in lysis buffer [25 mM Tris–phosphate pH 7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 0.5% Triton X-100] for 15 min. Cells were then scraped, transferred and spun at 15 000 g for 5 min. Supernatants were transferred and 20 l aliquots were assayed in duplicate for luciferase activity as described by Doyle (1996). Readings were taken for 2 s/sample in a Labsystems Lumoskan Luminometer. Chlorophenol red--D-galactopyranoside (CPRG) assays were carried out in duplicate for each condition using 30 l of cell lysate in 20 l (4 mg/ml) of CPRG (Calbiochem) and 150 l of CPRG buffer at 37°C for 12–18 h. Plates were read at 595 nm in a Labsystems Multiscan Platereader. Raw luciferase values were normalized for both transfection efficiency as determined by CPRG assay and cell number as determined by protein assay (Bio-Rad). Luciferase units are shown as (corrected) relative light units for a given condition performed in triplicate, with error bars indicating the standard deviation for that condition.

Protein expression

GST:RORLBD was transformed into BL21DE(3) competent cells (Novagen) and a 50 ml LB culture grown overnight in 100 g/ml ampicillin at 37°C. The next morning, the culture was diluted 1:20 in LB/ampicillin and grown at 37°C until OD 1.0, at which point isopropyl--D-thiogalactopyranoside (IPTG, 0.1 mM final concentration; Sigma) was added to the culture and grown for an additional 2 h at room temperature (20°C). The cells were pelleted and resuspended in lysis buffer [20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1.0 mM EDTA pH 8.0, 1.5% sarkosyl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT and 2 g/ml aprotinin, leupeptin and pepstatin] on ice. Cells were lysed via sonication, centrifuged at 15 000 g and the supernatant removed. Triton X-100 was added to a final concentration of 3.3%, and the supernatant was added to glutathione–Sepharose beads (Pharmacia). GST:RORLBD and GST alone were purified via affinity chromatography at 4°C, washed extensively in lysis buffer (containing 0.5% NP-40 instead of sarkosyl) and then dialyzed in 50 mM Tris–HCl pH 8.0. Proteins were volume reduced via ultrafiltration and stored in 10% glycerol in 50 mM Tris–HCl pH 8.0. Analysis via SDS–PAGE showed that the protein was purified to >95% homogeneity. His-tagged 1–117:P300 was purified via affinity chromatography (as described above) using Ni–NTA–agarose (Qiagen), lysis buffer (5 mM imidazole pH 7.4, 500 mM NaCl, 20 mM Tris–HCl pH 8.0, 0.1% NP-40 and protease inhibitors) and washing buffer (60 mM imidazole pH 7.4, 500 mM NaCl, 20 mM Tris–HCl pH 8.0). Recombinant 1–117:P300 was eluted (400 mM imidazole pH 7.4, 500 mM NaCl, 20 mM Tris–HCl pH 8.0), dialyzed and concentrated as described above. GST:CaMKI and maltose-binding protein (MBP):KKB were purified using similar affinity techniques to those previously described (Haribabu et al., 1995; Anderson et al., 1998).

Kinase assay

GST:RORLBD and 1–117:P300 were used as substrates for in vitro kinase assays with recombinant CaMKI and CaMKKB. Briefly, 500 ng of GST:RORLBD or 1–117:P300 were incubated in kinase buffer (50 mM Tris–HCl pH 8.0, 40 mM MgCl₂, 4 M CaM, 8 mM CaCl₂, 1 mM ATP, 0.1% Tween-20 and 2 Ci of [-³²P]ATP/reaction) in either the absence or presence of 200 ng of recombinant GST:CaMKI and/or 500 ng MBP:CaMKKB for 20 min at 30°C. The reactions were stopped by the addition of 2 SDS–PAGE loading buffer and fractionated on a 12% SDS–polyacrylamide gel, stained with Coomassie Blue dye and dried on Whatman 3MM paper. The gel was then exposed in a phosphorimager cassette for 8 h and imaged with a Molecular Dynamics PhosphorImager.

We are deeply indebted to V.Giguère and D.McDonnell for graciously supplying many of the plasmids used throughout the course of this study. We would also like to thank B.O'Malley, A.Cooney, E.Cocoran and C.-Y.Chang for providing reagents used in this work, as well as the members of both the Means and McDonnell laboratories for their many helpful comments and discussions. This work was supported in part by National Institutes of Health Grants HD07503 and GM33976 to A.R.M. and NRSA F32AI0976464 to C.D.K.

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