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J. Biol. Chem., Vol. 281, Issue 1, 9-12, January 6, 2006
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From the Sealy Center for Cancer Cell Biology and Department of Pharmacology, University of Texas Medical Branch, Galveston, Texas 7555-1048
Received for publication, June 28, 2005 , and in revised form, November 14, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein kinase A (PKA) was first identified as an inhibitory component of the Hh pathway in Drosophila (15–19). PKA fulfills its negative role by phosphorylating full-length Ci (Ci155) at several Ser/Thr residues, priming it for further phosphorylation by glycogen synthase kinase 3 (GSK3) and casein kinase I (CKI) (20–22). Hyperphosphorylation of Ci155 targets it for proteolytic processing to generate the repressor form (Ci75) (23). Consistent with this, overexpressing a constitutively active form of PKA catalytic subunit (PKAc), mC*, blocks Ci155 accumulation and Hh target gene expression (24). In addition to its inhibitory effects, PKA phosphorylation at the C terminus of SMO in Drosophila, but not in mammals, enhances hedgehog-mediated signaling (25, 26).
While the regulation of CI cleavage by PKA phosphorylation is well documented, very little is known about the role of PKA in Gli regulation. In vertebrates, there are three Gli molecules, Gli1, Gli2, and Gli3. Gli3 can be processed in a manner similar to CI, a process regulated by PKA (27, 28). The fact that Gli3 expression is often not detectable in human cancer suggests that Gli3 does not play a significant role in hedgehog-driven carcinogenesis (29–31). In contrast, Gli1 and Gli2 are expressed in tumors with activated hedgehog signaling (2, 29–36). Here, we report that the cAMP/PKA signaling axis regulates Gli1 protein localization, in part, through phosphorylation of Gli1 at a site near the nuclear localization signal (NLS). We propose that this unique regulation is an important mechanism by which PKA inhibits transcriptional activity of Gli molecules.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells with expression of Gli1 (with C-MYC tag) were treated with 10 µM H89 (Calbiochem) or 0.4 ng/ml leptomycin B (LMB, Sigma) for 8 h. For forskolin treatment (20 µM for 8 h), cells were pretreated with phosphodiesterase inhibitor IBMX (100 µM) for 30 min before addition of forskolin. Immunofluorescent detection of C-MYC-tagged Gli1 was performed as described previously with Cy3-conjugated MYC antibody 9e10 (Sigma) (1:100 dilution) (37). Gli1 localization was detected under a fluorescent microscope; the percentage of Gli1 in the nucleus or the cytoplasm was calculated for each experiment from over 200 Gli1-expressing cells, and the experiment was repeated three times.
Gli1 cDNA was generously provided by Dr. Bert Vogelstein and cloned into pCDNA3.1 with a C-MYC tag at the N terminus. Gli1-GFP construct was made by subcloning Gli1 cDNA into pEGFP-3C using BamHI (5') and NotI (3') sites. Point mutations of Gli1 were made by in situ mutagenesis in our DNA Recombinant Laboratory Core Facility or by PCR-based mutagenesis. All mutations were confirmed by sequencing of the entire coding region. Clones containing only the targeted mutations were used in the studies.
Immunoprecipitation and in Vitro Kinase Assay—Cells were lysed using Nonidet P-40 cell lysis buffer 48 h following Gli1 transfection. C-MYC-tagged Gli1 proteins were immunoprecipitated with an anti-MYC 9B11 antibody (Cell Signaling Inc.) for 3 h followed by incubation with A/G plus beads (Bethyl Laboratories, Inc.) for 1 h. The immunocomplexes were divided into two portions. One portion (20%) was separated by 10% SDS-PAGE and analyzed by Western blotting using an anti-MYC antibody 9B11. The remaining 80% of them were incubated with 0.6 ng of recombinant PKA catalytic subunit with ADBI buffer (20 mM MOPS, pH 7.2, 25 mM 
-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithionthreitol) containing 1 mg/ml bovine serum albumin, and 10 µCi of [
-32P]ATP at 30 °C for 30 min. The kinase reactions were terminated by adding 4x SDS sample buffer, and the samples were separated by 10% SDS-PAGE. Gels were dried on Whatman paper, followed by autoradiography. Western blotting analysis was performed according to a previously published procedure (37).
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-32P]ATP (3,000 Ci/mmol)], 250 µM Kemptide substrate in ADBI buffer at 30 °C for 10 min. Background was determined from reactions without substrate, and the total PKA activity was estimated in reactions containing 20 µM dibutyryl-cAMP. Aliquots were spotted onto Whatman P-81 paper, and the filters were washed in 0.75% phosphoric acid three times for 5 min per wash. 32P incorporation was determined by liquid scintillation counting. Protein concentration of the cell lysates was determined by a kit from Bio-Rad (Bio-Rad protein assay). Data are representative of three independent experiments.
Cell Fractionation—Following transfection, the COS7 cells were maintained in 10 cm cell culture dishes for 48 h. Before harvest, the cells were rinsed twice with cold phosphate-buffered saline, harvested by scraping with 1 ml of cold phosphate-buffered saline for each 10-cm dish, and collected by centrifugation at 3,000 x g for 30 s. One-fifth of the lysates were collected for detection of Gli1 expression by Western blotting. The cell pellets were incubated with 60 µl of buffer A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg of phenylmethylsulfonyl fluoride/ml, 1 µg of pepstatin A/ml, 1 µg of leupeptin/ml, 10 µg of soybean trypsin inhibitor/ml, 10 µg of aprotinin/ml, and 0.1% IGEPAL CA-630). After 10 min on ice, the lysates were centrifuged at 6,000 x g for 30 s at 4 °C. The supernatant fractions were saved as the cytoplasmic fraction. The pellet (containing the nuclei) was resuspended in buffer B (buffer A containing 1.0 M sucrose) and centrifuged at 15,000 x g for 10 min at 4 °C. The purified nuclei (pellets) were incubated in 30 µl buffer C (10% glycerol, 50 mM HEPES, pH 7.4, 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg of phenylmethylsulfonyl fluoride/ml, 1 µg of pepstatin A/ml, 1 µg of leupeptin/ml, 10 µg of soybean trypsin inhibitor/ml, 10 µg of aprotinin/ml) after vigorous vortex and centrifuged at 15,000 x g for 20 min at 4 °C, and supernatant fractions were collected. All extracts were normalized for protein amounts determined by Bio-Rad protein assay (Bio-Rad) and separated by 10% SDS-PAGE for further analysis (39). Antibodies to -tubulin (Sigma) and Lamin A/C (Santa Cruz Biotechnology Inc.) were used to detect the purity of cytoplasmic (-tubulin) and nuclear (Lamin A/C) fractions using Western blotting analysis.
Luciferase Reporter Gene Assay—For luciferase assay, NIH3T3 cells with stable expression of the Gli luciferase reporter were transfected with Gli1 constructs (0.5 µg/well) and the TK-Renilla control plasmid (5 ng/well) using Lipofectamine 2000. After 5 h, the medium was replaced with fresh growth medium and the cells were incubated in 5% CO2 at 37 °C overnight. Following treatment with forskolin or other compounds, the cells were harvested, and luciferase activity was measured with the dual luciferase reporter assay system (Promega) according to the manufacturer's instruction. In brief, the transfected cells were lysed in the 6-well plates with 100 µl of reporter lysis buffer and the lysate transferred into Eppendorf tubes. Cell debris was removed by centrifugation at top speed for 10 min in a microcentrifuge. 20 µl of the supernatant was mixed with 100 µl of buffer LAR II, and the absorbance was immediately measured (the first reading). After 3 s, 100 µl of Stop & Glo ® Reagent was added to measure the Renillar luciferase activity (the second reading). The value from the first reading was divided by the value from the second reading of each sample to obtain the luciferase activity. Each experiment was repeated three times with similar results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, we tested whether Gli1 protein localization can be altered by accumulation of the cellular cAMP level in COS7 cells. After transient transfection, the protein localization of Gli1 was detected by immunofluorescent staining of the C-MYC tag at the Gli1 N terminus and by cell fractionation. In the presence of 20 µM forskolin, which directly activates adenylyl cyclase and raises the cyclic AMP level (41), we observed that the percentage of cytoplasmic Gli1 was increased over 5-fold, whereas the percentage of nuclear Gli1 was reduced by 80% (Fig. 1, A and B). The effect seems to be direct because the change in Gli1 localization can be observed 20 min after forskolin treatment. Conversely, addition of PKA inhibitor H89 led to a shift of Gli1 localization to the nucleus (Fig. 1, A and B). As a consequence of forskolin treatment, the cellular PKA activity was increased 2-fold (Fig. 1C). Conversely, addition of H89 into the medium inhibited the cellular PKA activity by 70% (Fig. 1C). Thus, Gli1 localization was correlated with the cellular PKA activity. To confirm the data from the immunofluorescent staining, we performed cell fractionation analysis. As shown in Fig. 1B, more Gli1 were in the nuclear fraction following H89 treatment whereas forskolin caused an increase of cytoplasmic Gli1 (Fig. 1B). Furthermore, we monitored localization of Gli1-GFP fusion protein with a time-lapse microscope in the presence of H89 or forskolin. Forskolin retained Gli1-GFP in the cytoplasm whereas H89 promoted nuclear accumulation of this fusion protein (data not shown). All these data indicate that Gli1 localization can be regulated by modulating the cellular PKA activity.
Gli1 protein shuttling between the nucleus and the cytoplasm was interrupted by inhibition of nuclear export with LMB, a specific inhibitor for CRM1-mediated nuclear export, resulting in nuclear accumulation of all Gli1 proteins (Fig. 1, A and B), supporting that Gli1 localization is a dynamic process and is tightly regulated. Our data suggest that direct phosphorylation of Gli1 by PKA is responsible for regulation of Gli1 protein localization.
Sequence analysis predicts five putative PKA sites in Gli1. The sequence around these PKA sites is highly conserved among Gli proteins (Fig. 2A). Several point mutations of Gli1 were made to test Gli1 regulation by PKA (see the diagram in Fig. 2B). These mutations were made by in situ mutagenesis in our DNA Recombinant Laboratory Core Facility or by PCR-based mutagenesis.
Using these mutant constructs, we assessed Gli1 localization in cultured cells. We found that mutation at Thr374 (Gli1/T374V) significantly affects Gli1 localization (Fig. 2C). Furthermore, the response of Gli1/T374V to forskolin treatment was nearly diminished (in response to forskolin treatment, 5-fold increase in cytoplasmic protein for wild type Gli1 but only 40% increase for Gli1/T374V) (also see the supplemental figure). In contrast, the protein localization of Gli1/S544G and Gli1/S544G/S560A was not different from the wild type Gli1 (supplemental figure). A mutant Gli1 (Gli1/T374V/S544G/S560A) with triple mutations at the PKA sites behaved like Gli1/T374V (supplemental figure), indicating that Ser544 and Ser560 are not involved in regulation of Gli1 localization. On the other hand, a mutation at S640A had only slight effects on Gli1 protein localization in response to forskolin (supplemental figure). These data indicate that Thr374 is the major site responsible for Gli1 protein localization in cultured cells.
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-32P]ATP and recombinant PKA in test tube and found that Gli1 was highly phosphorylated by PKA in vitro (Fig. 2D, left panels, first lane). Gli1 with mutations of all five PKA sites (PKA
) could not be phosphorylated by PKA (Fig. 2D, left panels, right lane). To test whether the Thr374 site of Gli1 is phosophorylated by recombinant PKA, we used a Gli1 fragment containing only two PKA sites: Thr296 and Thr374. We found that this Gli1 fragment (1–514 aa) with a mutation at Thr374 was not able to be phosphorylated by recombinant PKA in vitro (Fig. 2D, center panels). Even a single point mutation at Thr374 significantly reduced PKA-mediated phosphorylation of full-length Gli1 (Fig. 2D, right panels), indicating that the Thr374 site is a major PKA site of Gli1 phosphorylation. In addition, our data also suggested that Ser544, Ser560, and Ser640 can be phosphorylated by PKA in vitro (data not shown here). The above data indicate that Thr374 is a major PKA site involved in regulation of Gli1 protein localization. In the three-dimensional structure, Thr374, together with the adjacent Asp375, is close to the first basic residue cluster (Arg380/Lys381/His382) of the bipartite motif (the classic NLS) in Gli1 (Fig. 3A) (42). It is known that protein phosphorylation at the residue next to the bipartite motif inhibits its binding affinity to importins, leading to reduced nuclear localization of the target protein (43). We predict that phosphorylation of Thr374 will increase the local negative charge, leading to reduced NLS functions and accumulation of Gli1 in the cytoplasm. Indeed, Gli1/T374D was preferentially localized to the cytoplasm (Fig. 3B). Conversely, Gli1/T374K has a high local positive charge near the NLS, and we found that Gli1/T374K predominantly localized to the nucleus (Fig. 3B). Localization of these Gli1 proteins was further confirmed by cell fractionation (Fig. 3B). These data support our hypothesis that one mechanism by which the cellular PKA activity regulates Gli1 localization is through altering the local charge nearby the NLS of Gli1.
If Gli1 nuclear localization is regulated by PKA phosphorylation through a NLS-dependent mechanism, disruption of the NLS should affect localization of these mutant Gli1 molecules. As shown in Fig. 3C, we found that Gli1/K381E, which is predicted to disrupt the NLS, was predominantly localized to the cytoplasm, confirming the role of NLS in Gli1 localization (12). Although Gli1/T374K localizes predominantly to the nucleus, additional mutation at K381 (K381E) retained Gli1/T374K to the cytoplasm (Fig. 3C), suggesting that regulation of Gli1 localization by T374 phosphorylation requires the intact NLS.
The ultimate effect of Gli1 is transcriptional activation of the downstream target genes. To assess whether Gli1 localization affects its transcriptional activity, we established stable expression of Gli1 luciferase reporter under the control of Gli responsive elements in NIH3T3 cells (13). By measuring the reporter luciferase activity, we examined the association of Gli1 localization with its transcriptional activity (Fig. 3D). If Gli1/K381E remains preferentially in the cytoplasm, the transcriptional activity was low. In contrast, Gli1 with a defective NES (Gli1/L496V/L498V), which localizes predominantly in the nucleus, was more active (Fig. 3D). Thus, the Gli1 luciferase reporter activity in these cells is very sensitive to Gli1 localization. We examined the effects of forskolin to Gli1-mediated transcriptional activity in this NIH3T3 stable cell line. Like the wild type Gli1, Gli1/T374V can activate the Gli luciferase reporter (Fig. 3E). Consistent with its cytoplasmic localization, the luciferase activity in cells expressing the wild type Gli1 was reduced after forskolin treatment (Fig. 3E). In contrast, Gli1/T374V-mediated reporter gene activity was not affected by forskolin (Fig. 3E). These data suggest that Thr374 is an important PKA site responsible for PKA phosphorylation and for the transcriptional activity of Gli1.
Based on these data, we proposed a mechanism by which the cAMP/PKA signaling axis mediates regulation of Gli1 localization. Gli1 enters the nucleus through a nuclear localization signal. With the accumulation of cAMP in the cell, Thr374 gets phosphorylated. Phosphorylation of Thr374 will increase the local negative charge nearby the NLS, which results in inhibition of NLS function. Consequently, Gli1 is retained in the cytoplasm and is unable to activate the target genes. Since this Thr residue is highly conserved among Gli proteins, we anticipate that the same mechanism is applicable to PKA regulation of other Gli molecules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although our studies demonstrated that Thr374 is a major site for PKA-mediated regulation of Gli1 localization (Figs. 2 and 3 and supplemental figure), mutation at this site did not completely abolish the effects of forskolin (supplemental figure). To identify an additional site required for this regulation, we made double and triple point mutations of Gli1 at the PKA sites (Fig. 2B). Our data indicate that mutations at Thr374 and Ser640 completely abolished the response to forskolin treatment, indicating that Ser640 is another site involving PKA-mediated regulation of Gli1 localization (supplemental figure). However, mutation at Ser640 alone had no effect on Gli1 protein localization and had only a slight effect in response to forskolin treatment, suggesting that Ser640 is not a primary site for Gli1 regulation. This hypothesis was further supported by the fact that Gli1/S640E and Gli1/S640R, unlike Gli1/T374D and Gli1/T374K, did not alter Gli1 protein localization (data not shown here). Thus, we believe that additional structural information of Gli1 near the Ser640 region is required to understand the molecular mechanism by which PKA phosphorylation at Ser640 affects Gli1 localization.
Further studies of Gli1 phosphorylation can be facilitated by measuring the stoichiometry of phosphorylation for Gli1. Currently, a large amount of purified Gli1 protein is not available, making it difficult to calculate the stoichiometry of phosphorylation for such a large protein (150 kDa for Gli1). This issue may be addressed in the future using highly purified Gli1 fragments from bacteria.
In addition, our data indicate that Ser544 and Ser560 residues are not involved in regulation of Gli1 protein localization. It will be interesting to know how these two sites are involved in regulation of Gli1 functions.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.
1 To whom correspondence should be addressed: Sealy Center for Cancer Cell Biology, MRB 9.104, UTMB, 301 University Blvd., Galveston, TX 77555-1048. Tel.: 409-747-1845; Fax: 409-747-1938; E-mail: jinxie{at}utmb.edu.
2 The abbreviations used are: Hh, hedgehog; PTC, patched; SMO, smoothened; PKA, protein kinase A; CI, cubitus interruptus; Shh, sonic hedgehog; NLS, nuclear localization signal; NES, nuclear export signal; aa, amino acid; LMB, leptomycin B; IBMX, isobutylmethylxanthine; GFP, green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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