Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3

Abstract

Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. Using a tandem affinity purification (TAP) strategy and mass spectrometry, we have found that Gli1 interacts with 14-3-3epsilon, and that Gli2 and Gli3 also bind to 14-3-3epsilon through homologous sites. This interaction depends on their phosphorylation, and cAMP-dependent protein kinase (PKA), a known negative regulator of Hedgehog signaling serves as a responsible kinase. A Gli2 mutant engineered to eliminate this interaction exhibited increased transcriptional activity (2 approximately 3x). Transcriptional repression by 14-3-3 binding was also observed with Gli3, when its N-terminal repressor domain was deleted. The phosphorylation sites responsible for the binding to 14-3-3 are distinct from those required for proteolysis, the known mechanism for PKA-induced repression of Hh signaling. Our data propose a novel mechanism in which PKA down-regulates Hedgehog signaling by promoting the interaction between Gli and 14-3-3 as well as proteolysis. Given the certain neuronal or malignant disorders in human caused by the abnormality of 17p13 encompassing 14-3-3epsilon overlap with increased Hh signaling, the Gli-14-3-3 interaction may have pathological significance for those human diseases.

FIGURE 1.

Identification of 14-3-3ϵ as a Gli1-interacting protein. A, schematic representation of the strategy for TAP using an MEF tag (10, 11). The pcDNA3 vector is designed to express a protein with tandem Myc and FLAG tags at the N terminus of the bait protein. The first round of affinity purification is performed using anti-Myc Ab, followed by cleavage with TEV protease. The second round of affinity purification is performed using anti-FLAG Ab, followed by specific elution with FLAG peptide. The eluted complex was separated by SDS-PAGE and silver-stained. Specific bands were digested with trypsin and analyzed by LC-MS/MS. B, after lysis of pcDNA3-MEF-Gli1- or pcDNA3-MEF-transfected 293T cells, the expressed Gli1 was recovered using the MEF TAP procedure. The proteins bound to Gli1 were eluted from the beads, separated by SDS-PAGE (10/20%), and silver-stained. The molecular masses of the protein are indicated. The arrowhead indicates the position of Gli1. The four protein bands, indicated by arrows, were excised from the gel and subjected to in-gel tryptic digestion. LC-MS/MS analysis identified the proteins as 14-3-3ϵ, semenogelin I, and 70-kDa heat-shock proteins 5, 6, and 8. C, lysates from PLC/PRF/5 cells were subjected to immunoprecipitation using anti-Gli1 Ab or control IgG followed by Western blotting using anti-Gli1 (upper blot) and anti-14-3-3ϵ (lower blot) Abs to detect endogenous interaction. D, immunoprecipitation using anti-14-3-3ϵ Ab was performed as described in C.

FIGURE 2.

Interaction of Gli1 with 14-3-3 through Ser-640. A, Gli1 interacts with 14-3-3ϵ but not with the K49E 14-3-3ϵ mutant, which exhibited no affinity for the phosphorylated sequence. FLAG-tagged Gli1 was co-expressed with Xpress-tagged wt or K49E 14-3-3ϵ in 293T cells. Cell lysates were subjected to immunoprecipitation using monoclonal anti-FLAG Ab followed by Western blotting using monoclonal anti-Xpress Ab to detect associated 14-3-3ϵ (upper blot) or using anti-FLAG Ab to monitor the efficiency of Gli1 protein expression (middle blot). Lysate samples were also subjected to immunoblot analysis with anti-Xpress Ab (lower blot). B, Scansite prediction of the 14-3-3 binding site on Gli1. Mode1 and mode2 are consensus sequences for the 14-3-3 binding site. The amino acid sequences containing Ser-545, -640, -659, and -963 were predicted to bind to 14-3-3. C, Ser-640 of Gli1 is important for the interaction with endogenous 14-3-3. FLAG-tagged Gli1 proteins engineered with a Ser → Ala mutation were expressed in 293T cells. Immunoprecipitation of the cell lysates was performed with anti-FLAG Ab, followed by Western blotting using polyclonal anti-14-3-3 Ab (upper blot) or anti-FLAG Ab (middle blot). Lysate samples were also blotted using anti-14-3-3 Ab as a control (lower blot).

FIGURE 3.

Interaction of Gli2 and Gli3 with 14-3-3 through the conserved site phosphorylated by PKA. A, schematic drawing of transcription factor Gli and conservation of its 14-3-3 binding site. Repressor, N-terminal repressor domain; ZF, zinc finger; NLS, nuclear localization signal; NES, nuclear export signal; AD, transactivation domain. Other conserved regions are indicated in black. B, conserved sequences of Gli1, Gli2, and Gli3 were phosphorylated by PKA in vitro. Kinase assays using recombinant PKA were performed with GST (about 26 kDa) or a GST fusion protein containing Gli1 Ser-640, Gli2 Ser-956, or Gli3 Ser-1006 and their respective flanking sequences (about 35, 40, or 38 kDa, respectively), in both wt and Ser → Ala-substituted forms. Reactions were resolved by SDS-PAGE, and the gel was stained with Coomassie Blue (right panel), dried, and analyzed by autoradiography (left panel). C, interaction of Gli2 with endogenous 14-3-3 through Ser-956. FLAG-tagged wt and mutant Gli2 (S956A or S975A) were expressed with PKA-CA in 293T cells. Cell lysates were immunoprecipitated using anti-FLAG Ab and blotted using anti-14-3-3 Ab (upper blot) or anti-FLAG Ab (middle blot). Samples of the lysates were blotted with anti-14-3-3 Ab as a control (lower blot). D, interaction of Gli3 with endogenous 14-3-3 through Ser-1006. A similar assay was performed with FLAG-tagged wt or mutant Gli3 (S1006A and/or S1026A). Wild-type and S1026A Gli3 interacted with 14-3-3, but S1006A and S1006A/S1026A mutants lost their affinity for 14-3-3. E, phosphorylation of Gli2 Ser-956 in 293T cells increased with FSK exposure, and the affinity of Gli2 for 14-3-3 increased as this phosphorylation increased. The 293T cells were transfected with a FLAG-tagged wt or S956A Gli2 vector or the empty vector 24 h before addition of 40 μm FSK to the growth medium. The cells were lysed after 0, 1, 2, and 4 h of FSK exposure. Lysates were subjected to immunoprecipitation using anti-FLAG Ab and blotted with Abs specific for the 14-3-3 binding site (RXXpSXP), FLAG, or 14-3-3. Samples of the lysates were blotted using anti-14-3-3 Ab as a control.

FIGURE 4.

PKA-induced interaction with 14-3-3 attenuates the transcriptional activity of Gli2 and ΔN-Gli3, a Gli3 construct lacking its N-terminal repressor domain. A, 293T cells were co-transfected with Gli-BS-Luc, pRL-SV40 (internal control), PKA-CA, and a Gli1, Gli2, Gli3, or ΔN-Gli3 expression plasmid, and the luciferase activity of the wt and 14-3-3 binding site mutant Gli proteins were compared. White bar, wt; black bar, mutant. Wild-type Gli2 and ΔN-Gli3 were less active than their respective Ser → Ala mutants. B, transcriptional activity of Gli3 was almost equal to that of the empty vector, and deletion of the N-terminal repressor domain to create ΔN-Gli3 restored the transcriptional activity. The recovered activity of the constructs with the capacity to interact with 14-3-3 (wt and S1026A) was increased by the S1006A 14-3-3 binding site mutation (mutant S1006A and double-mutant S1006A/S1026A). C, Gli2 P958A mutant also lost the capacity to interact with 14-3-3, as shown by immunoprecipitation with anti-FLAG Ab and immunoblotting. Phosphorylation status could not be confirmed; however, since the proline residue at +2 relative to the putative pSer is required for detection by the anti-14-3-3 binding site Ab. D, transcriptional activity of Gli2 constructs was measured as described in A and B. The activities of the P958A and S956A Gli2 mutants were similar. E, in mutant ΔPKA-Gli2, the four regions of Gli2 containing PKA consensus sites responsible for its degradation following ubiquitination were deleted. The effect of interaction with 14-3-3 on transcriptional activity was not affected by these mutations. The data shown are representative of three independent experiments performed in duplicate (error bars). **, p < 0.01; *, p < 0.05.

FIGURE 5.

Interaction of Gli2 with 14-3-3 does neither affect its subcellular localization, SUFU-binding activity, nor the affinity for target oligonucleotide. A, neither PKA-mediated phosphorylation nor 14-3-3 binding relocalize Gli2 into the cytosol. Immunofluorescence pictures of representative HeLa cells show that both wt and S956A Gli2 localize in the nucleus in FSK-treated cells. The Myc-tagged Gli2 proteins are immunostained (green); the nuclei are stained with propidium iodide (red). Scale bar, 20 μm. B, co-expression of SUFU, which represses the transcriptional activity of both wt and S956A Gli2 and eliminates the effect of interaction with 14-3-3. The data shown are representative of three independent experiments performed in duplicate (error bars). **, p < 0.01; *, p < 0.05. C, immunoprecipitation using a lysate from cells co-expressing Myc-tagged SUFU, a FLAG-tagged Gli2 construct, and PKA-CA proves that 14-3-3 binding does not influence the interaction of Gli2 with SUFU. D, binding of Gli2 to target oligonucleotide. Cells were transiently co-transfected with expression plasmids encoding PKA-CA and FLAG-tagged wt or S956A Gli2 or the empty vector. Cell lysates were immunoprecipitated using anti-FLAG Ab, and bound proteins were eluted using 3× FLAG peptide. Eluted lysates were subjected to electrophoretic mobility shift assay. Both wt and S956A mutant Gli2 formed specific complexes with the consensus (labeled) Gli-binding oligonucleotide (arrow). Addition of a 100-fold excess of unlabeled Gli outcompeted the labeled oligonucleotide.

FIGURE 6.

FSK suppresses the expression of Gli1 and depletion of 14-3-3ϵ cancels the suppression in PLC/PRF/5 and NIH3T3. A, cell lysates from PLC/PRF/5 cells, treated overnight with DMSO or 40 μm FSK, were subjected to immunoblot analysis. FSK treatment reduced Gli1 expression in control cells, but this effect was canceled in 14-3-3ϵ knockdown cells. 14-3-3ϵ knockdown was corroborated using anti-14-3-3ϵ and anti-α-tubulin Abs (α-tubulin: loading control). B, normalized Gli1 and Ptch1 mRNA levels in SAG ± FSK-induced NIH3T3 cells were determined by quantitative PCR. Cells were treated for 30 h with DMSO or 100 nm SAG ± 1 μm FSK in medium with 0.5% BS. C, Gli1 protein level was determined by immunoblotting. Cell lysates from NIH3T3 cells, treated as A, were subjected. Immunoblot using anti-phospho PKA substrate Ab showed efficient PKA phosphorylation induced by FSK (α-tubulin: loading control). D, cell lysates from 14-3-3ϵ knockdown NIH3T3 cells, treated with SAG ± FSK, were subjected to immunoblotting to determine Gli1 protein levels. 14-3-3ϵ knockdown was corroborated using anti-14-3-3ϵ and anti-α-tubulin Abs (α-tubulin: loading control). E, NIH3T3 cells were co-transfected with Gli-BS-Luc, pRL-SV40 (internal control), and 14-3-3 inhibitor EYFP-difopein or its control EYFP-R18Lys expression vector. After treatment as A, luciferase activity were determined. The data shown are representative of three independent experiments performed in duplicate (error bars). **, p < 0.01; *, p < 0.05.