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Previous Article | Next Article 
Molecular and Cellular Biology, August 2006, p. 5784-5796, Vol. 26, No. 15
0270-7306/06/$08.00+0 doi:10.1128/MCB.00232-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Joanna Lisztwan ,
,¶
Claudio R. Thoma,¶
Christiane Wirbelauer,
Robert E. Barry,|| and
Wilhelm Krek*
Institute for Cell Biology, ETH Zurich, 8093 Zurich, Switzerland
Received 8 February 2006/ Returned for modification 13 March 2006/ Accepted 14 May 2006
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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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VHL disease demonstrates a complex genotype-phenotype relationship suggesting the operation of distinct tumor suppressor mechanisms. Indeed, pVHL, through its oxygen-dependent polyubiquitylation of HIF
, has been shown to play a central role in the mammalian oxygen-sensing pathway (9, 16, 18, 19, 31). However, a distinct aspect of pVHL's tumor suppressor function has previously been revealed through studies demonstrating a HIF (hypoxia-inducible factor)-independent functional association of pVHL with the microtubule (MT) apparatus (14). The form of pVHL most prominently associated with MTs in vivo appears to be the long form of pVHL, pVHL30, and not its short form, pVHL19 (14). pVHL19 is mostly found in the nucleus; however, cytoplasmic pVHL19 can bind to and stabilize MTs (14). Functional analysis of naturally occurring pVHL mutants revealed a link between altered MT stabilization function and pVHL-associated tumor-suppressing activity. In keeping with these findings, the MT-stabilizing activity of pVHL has been shown to be localized specifically to the cell periphery (29). Thus, apart from its role in oxygen sensing, pVHL also participates in the control of MT dynamics.
Here we analyzed the regulation of pVHL's MT-stabilizing activity to gain further insight into this potential tumor suppressor activity. Our data show that the functional association of pVHL30 with MTs is dynamically regulated by a dual kinase mechanism. A priming phosphorylation of pVHL30 on S72 allows phosphorylation at S68 by glycogen synthase kinase 3 (GSK3), thereby negatively regulating pVHL-mediated MT stabilization. We also provide data suggesting that phosphorylation of pVHL on S68 and S72 affects not only pVHL's MT-stabilizing activity but also the interaction of pVHL with HIF.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of plasmids.
pcDNA3-HA-VHL deletion constructs have been described earlier (14), except for pcDNA3-HA-VHL(
54-73) and pcDNA3-HA-VHL1-195. All mutants of pVHL were generated by a two-step PCR-based mutagenesis procedure using pcDNA3-HA-VHL30 as a template. Individual PCR products were subsequently digested with BamHI and EcoRI and cloned into a pcDNA3 derivative containing a hemagglutinin (HA) epitope. To obtain pGST-VHL30 mutants, the corresponding fragments were subcloned into pGEX-4T-1 (for bacterial expression) or pAc-GST (for expression in Sf9 cells) using BamHI and EcoRI. GSK3ß constructs were also generated by a two-step PCR-based mutagenesis procedure using pMT-GSK3ß as a template. For the generation of retrovirus expression plasmids pCMV(neo-retro)-VHL30 and the indicated mutants thereof, the corresponding fragments were subcloned into pCMV(neo-retro) using BamHI and XhoI restriction sites. The VHL targeting and nonsilencing microRNA 30-based short-hairpin RNA (shRNAmir) were obtained from Open Biosystems (Huntsville, AL). Clones V2HS_201603 and RHS1703 were cloned into pLMP (7) as EcoRI/XhoI fragments. All constructs were confirmed by sequence analysis. Details on the generation of constructs are available upon request.
Generation of antibodies.
Generation and purification of anti-pVHL and anti-Cul2 antibodies have been described previously (14, 25). Phosphospecific antibodies were raised against the synthetic peptides VLRSVNSpREPSpQVIF and SVNSREPSpQVIFCNR, corresponding to phosphorylated S68 or S72 of human pVHL, respectively. Before injection into rabbits, the peptides were coupled to keyhole limpet hemocyanin (Pierce) by glutaraldehyde coupling. Polyclonal rabbit sera were either preabsorbed with unphosphorylated pVHL peptide (VLRSVNSREPSQVIFCNR) to obtain an anti-pVHL(S72-P) antibody or preincubated with 20 µM unphosphorylated pVHL peptide and S72-phosphorylated peptide (SVNSREPSpQVIFCNR) to obtain an anti-pVHL(S68-P) antibody. A rat monoclonal anti--tubulin (YL1/2)-producing cell line was from ATCC. Anti-HA antibodies were from Santa Cruz (Y-11), Babco (12CA5), and Roche (3F10). Anti-GSK3ß, anti-Cdk2, anti-pVHL (monoclonal antibody Ig32), anti-glutathione S-transferase (anti-GST) (G-7781), and anti-acetyl-tubulin (6-11B-1) were from Transduction Laboratories, Santa Cruz Biotechnology, Inc., Pharmingen, and Sigma, respectively. Anti-HIF2 and anti-Glut-1 were obtained from Novus Biologicals.
Immunoblotting, immunoprecipitation, immunofluorescence microscopy, and in vitro HIF2/pVHL binding assay.
Immunoblotting, immunoprecipitation, and immunofluorescence experiments were performed essentially as described previously (14, 26). Immunoblots were processed by ECL (Amersham) according to the manufacturer's instructions. To test the interaction of pVHL with HIF2 in vitro, GST-pVHL30 was purified from programmed Sf9 lysates. Purified protein was incubated in the absence or presence of 50 U of recombinant GSK3 (New England Biolabs) in 30 µl of GSK3 kinase buffer (20 mM Tris, 10 mM MgCl2, 5 mM dithiothreitol [DTT] at pH 7.5) plus ATP. Kinase reactions were performed for 30 min at 30°C. Then beads were washed four times with 1 ml of TNN buffer (50 mM Tris, 250 mM sodium chloride, 5 mM EDTA, 0.5% NP-40, 50 mM sodium fluoride, 0.2 mM ortho-vanadate, 1 mM DTT, 10 mM phenylmethylsulfonyl fluoride, and 1 µg ml–1 aprotinin at pH 7.5), before GST-pVHL30 was incubated with cold in vitro-translated, HA-tagged HIF2 in 0.5 ml of TNN buffer for 2 h. Finally, beads were washed four times with 1 ml of TNN buffer, and bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting. To produce HA-tagged HIF2, plasmid DNA was translated in vitro using the TNT Quick Coupled transcription/translation system (Promega).
In vitro phosphorylation of recombinant pVHL.
GST-pVHL30 was expressed in Escherichia coli and purified using glutathione-Sepharose affinity resin (Sigma). Alternatively, GST-pVHL30 was purified from programmed Sf9 lysates. Purified protein was incubated with either 100 U of recombinant casein kinase I (CKI) (New England Biolabs) in 30 µl of CKI kinase buffer (50 mM Tris, 10 mM MgCl2, 5 mM DTT at pH 7.5) or 50 U of recombinant GSK3 (New England Biolabs) in 30 µl of GSK3 kinase buffer plus 10 µCi of [
-32P]ATP (2,000 Ci/mmol; Amersham). Kinase reactions were performed for 30 min at 30°C. To test whether pVHL is a primed substrate, GST-pVHL30 was first incubated with 
-phosphatase (100 U; New England Biolabs) for 30 min at 30°C and then washed with TNN buffer before the kinase assay was carried out. For sequential phosphorylation, purified proteins were incubated with recombinant CKI using unlabeled ATP. Then beads were washed four times with 1 ml of TNN buffer and twice with 1 ml of GSK3 kinase buffer before a kinase experiment with GSK3 was performed using radiolabeled ATP. Kinase reaction mixtures with protein kinase A (PKA) (500 U; New England Biolabs) were incubated as described above. Phosphorylated proteins were separated by SDS-PAGE, and gels were dried and visualized by autoradiography.
GSK3 peptide kinase assay.
In a total volume of 30 µl kinase buffer (20 mM Tris, 10 mM MgCl2, 5 mM DTT at pH 7.5) containing 100 µM [-32P]ATP (
1,000 cpm/pmol), 1 mM substrate peptide (VLRSVNSREPSQVIFCNR or SVNSREPSpQVIFCNR) and GSK3 (50 U; New England Biolabs) were incubated at 30°C for the indicated times. Reactions were stopped by addition of EDTA, pH 8.0, and 20 µl of supernatants was spotted onto squares of P-81 phosphocellulose paper (Whatman). These were then washed four times, for 10 min each time, in 1% phosphoric acid and once in acetone before being counted in a liquid scintillation counter. Experiments were performed in triplicate.
Microtubule cosedimentation and stability assays.
The cosedimentation assay has been described elsewhere (14). The MT stabilization assay was performed as described previously (14). Briefly, exponentially growing COS-7 cells were plated on coverslips and transfected the next day with the indicated constructs. Eighteen to 24 h posttransfection, cells were treated with 10 µM nocodazole for 20 min before being processed for immunofluorescence using anti--tubulin (YL1/2), anti-HA (Y11), and anti-GSK3ß antibodies. At least 300 cells per experiment were analyzed for an intact MT network. Only cells with intact nuclei and a clearly detectable cytoplasmic signal of HA-pVHL were included in the evaluation. Experiments were repeated as blind assays.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-phosphatase prior to being tested as a GSK3 substrate (Fig. 2B). Dephosphorylated GST-pVHL30 served as a weak in vitro substrate for GSK3 in comparison to untreated protein (Fig. 2B, lanes 4 and 5). In agreement with this finding, bacterially expressed GST-pVHL30 did not serve as a GSK3 substrate (Fig. 2D, lane 1). These data suggests that phosphorylation of pVHL by GSK3 in vitro requires priming phosphorylation on one or more sites.
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To examine pVHL phosphorylation further, we raised antibodies that recognize specifically pVHL phosphorylated on S68 [anti-pVHL(S68-P)] or S72 [anti-pVHL(S72-P)] (Fig. 2E). Anti-pVHL(S68-P) antibody displayed a specific signal only when CKI-prephosphorylated GST-pVHL30 was incubated with GSK3 (Fig. 2F, lane 4), whereas anti-pVHL(S72-P) antibody detected selectively species phosphorylated by CKI (Fig. 2F, lanes 2, 4, and 5). Mutations at S68 or S72 of pVHL blocked the corresponding signals (Fig. 2F, lanes 5 and 6), suggesting that these antibodies recognize the relevant phosphorylated sites. In addition, a peptide encompassing serine residues 68 and 72 of pVHL was phosphorylated by GSK3 only when it was phosphorylated at S72 (Fig. 2G). These data fully support the above-noted observation that S68 phosphorylation of pVHL by GSK3 is dependent on phosphorylation at S72.
GSK3 phosphorylates pVHL30 on serine 68 in vivo. Given the specificity of our anti-phospho-antibodies in vitro (Fig. 2E and F), we next addressed their specificity in vivo (Fig. 3). Anti-pVHL(S68-P) antibody readily detected pVHL upon coexpression with functional GSK3ß and did not detect a specific signal when catalytically inactive GSK3ß(K85R) was coexpressed or pVHL30(S68A) was used (Fig. 3A, lanes 2 to 4). Anti-pVHL(S72-P) antibody did not detect a specific signal even when CKI and/or GSK3ß were coexpressed with pVHL (data not shown). These results imply that the anti-pVHL(S68-P) antibody signal is specific and a consequence of pVHL phosphorylation by GSK3 in vivo. Similar experiments carried out with anti-pVHL(S72-P) revealed that this antibody is not suitable for in vivo studies due to limitations in affinity and specificity.
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Human cells express two pVHL isoforms, pVHL30 and pVHL19 (4, 17, 36), but the function of only one, namely, pVHL30, has been linked to pVHL's MT stability control (14). Strikingly, pVHL30 was phosphorylated on S68 by GSK3ß and pVHL19 was not, although both pVHL species and GSK3ß were expressed to similar levels (Fig. 3D, lanes 2 and 4). This suggests that only pVHL30, the MT-associated pVHL species (14), is targeted for phosphorylation by GSK3ß in vivo. Notably, this finding is in full agreement with a recent report showing that pVHL30, but not pVHL19, is phosphorylated in mammalian cells (28). Of note, GSK3 also failed to phosphorylate recombinant GST-pVHL19 in vitro (data not shown), most likely because CKI could not prephosphorylate GST-pVHL19 (see Fig. S1B in the supplemental material).
Human primary or cancer cells express pVHL19 abundantly, but little pVHL30 (14), thus making it difficult to determine whether native pVHL30 is phosphorylated on S68 in vivo. To circumvent this limitation, we used the mouse renal cancer cell line IMCD-3, which expresses higher levels of pVHL25 than of pVHL21, the long and short isoforms in mice (Fig. 3E, bottom). Therefore, taking into account that the primary sequence of mouse and human pVHL is 100% identical in the region encompassing S68 and S72 (whereas S34 and S38 of mouse pVHL correspond to S68 and S72 of human pVHL), this mouse cell line allowed us to address the phosphorylation of endogenous pVHL by GSK3 (Fig. 3E). As shown in Fig. 3E, immunoblotting of anti-pVHL(m)CT immunoprecipitates derived from IMCD-3 cells subjected to serum deprivation, a condition known to activate endogenous GSK3, with anti-pVHL(S68-P) antibody revealed a specific phospho-signal. LiCl treatment reduced the signal significantly (Fig. 3E; compare lanes 3 and 2), implying that the anti-pVHL(S68-P) antibody immune reactivity is dependent on active GSK3. No such signal was seen in control immunoprecpitates (Fig. 3E, lanes 1 and 4). Moreover, reblotting of the same membrane with a monoclonal antibody against pVHL demonstrated the identity of the phospho-signal as phosphorylated pVHL25 (Fig. 3E, center). These results would suggest that mouse pVHL25 is phosphorylated on S34 (the residue corresponding to human S68) in a GSK3-dependent manner in vivo. Since the band corresponding to the pVHL21 isoform comigrated with an unspecific band that appeared in these immunoprecipitation/immunoblotting experiments, we were unable to assess whether pVHL21 is phosphorylated on this site.
GSK3 negatively affects pVHL's MT stabilization activity. GSK3ß has been shown previously to regulate MT dynamics via phosphorylation of MT-associated proteins (5, 12, 13, 30, 35, 39, 43), including the tumor suppressor protein adenoma polyposis coli (APC) (10, 44). Therefore, we tested whether GSK3ß would affect pVHL30's MT stabilization function. To this end, we adapted a previously developed assay (14). COS-7 cells expressing either HA-tagged pVHL30 alone or HA-pVHL30 and GSK3ß were treated with nocodazole, and transfected cells were assessed for the presence of stable MTs by using triple immunofluorescence microscopy (Fig. 4A). As shown previously (14), expression of pVHL30 protected MTs from depolymerization. However, coexpression of GSK3ß with pVHL30 inhibited this activity (Fig. 4A). Addition of LiCl, a known GSK3 inhibitor (23, 37), blocked the negative effect of GSK3ß, and coexpression of GSK3ß(K85R) failed to inhibit pVHL's MT stabilization function (Fig. 4A). Of note, addition of 20 mM LiCl alone did not lead to a significant increase of cells displaying stabilized MTs, which is in full agreement with previous findings (24). These results, together with the quantification of GSK3ß's effect on pVHL's MT stabilization function (Fig. 4B), imply a role for GSK3ß in the negative regulation of pVHL30's MT stabilization function.
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To address whether GSK3 might alter the subcellular localization of pVHL species, we either added LiCl to cells to inhibit endogenous GSK3 or overexpressed GSK3ß and analyzed the localization of endogenous pVHL in transfected cells by immunofluorescence microscopy (Fig. 5). Regardless of whether chemical inhibitors were used or not, the majority of pVHL30 was concentrated on the MT organizing center in close proximity to the nucleus (Fig. 5A). Upon LiCl treatment, a subfraction of pVHL30 was enriched in the cell periphery at sites of MT stabilization as a consequence of GSK3 inhibition. However, this result is not surprising, considering that chemically induced MT stabilization is known to affect the localization of pVHL30 (14). The overexpression of GSK3ß also did not obviously change the subcellular localization of native pVHL30, since the expression of wild-type or kinase-dead GSK3ß did not affect the accumulation of pVHL on the MT organizing center (Fig. 5B). Altogether, it appears that GSK3 does not grossly alter the localization of endogenous pVHL30.
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(10) resulted in similar effects on the MT stabilization function of pVHL30(S72D) (data not shown). Of note, pVHL mutants were expressed at similar levels (Fig. 8C), thus excluding the possibility that our results are a consequence of unequal expression. Finally, to test whether the pVHL30(S72D) mutation is actually mimicking phosphorylation at this site in vivo, as suggested by the data above, cells expressing pVHL30(S72D) were analyzed by immunoblotting (Fig. 8D). pVHL30(wt) or the S68A S72D mutant showed no obvious increase in phosphorylation of S68 (Fig. 8D, lanes 1, 4, and 5). However, pVHL30(S72D) was phosphorylated on S68 in a GSK3-dependent manner (Fig. 8D, lanes 2 and 3). Inactivation of GSK3 by a second inhibitor had similar effects (data not shown), further emphasizing the point that S72D substitution renders pVHL a GSK3 substrate in vivo. These data provide a potential explanation for why pVHL30(S72D) was altered in its MT stabilization function only under conditions where endogenous GSK was active, namely, because the S72D mutation allows for the efficient phosphorylation of pVHL30 by endogenous GSK3.
Considering that only a certain subset of reported pVHL mutations affect pVHL's MT-stabilizing activity (14), we speculated that these mutants do not stabilize MTs due to increased phosphorylation by GSK3. However, we did not observe any significant difference with regard to S68 phosphorylation when comparing these mutants with wild-type protein (see Fig. S2 in the supplemental material), ruling out the possibility that the reported decrease in MT-stabilizing activity by these pVHL mutants is a result of altered phosphorylation on S68.
Phosphomimicking mutations on S68 and S72 of pVHL30 negatively affect HIF2 binding.
Since Etienne-Manneville and Hall reported that the MT-dependent migration of cells can be regulated by GSK3 (10), we generated cell lines that stably express various pVHL30 derivatives in VHL-deficient 786-O cells (Fig. 9A) and analyzed their cell migration abilities. However, we could not observe a statistically significant difference with respect to cell migration when testing these various cell lines (data not shown). This finding is not surprising, given the fact that the migration of parental 786-O cells was not affected by nocodazole (data not shown). Thus, the migration of 786-O cells appears to be independent of MT dynamics.
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levels, we determined HIF2 protein levels in these cell lines (Fig. 9A). Regulation of HIF1 protein levels could not be addressed, since 786-O cells do not express HIF1 (31). Interestingly, pVHL30(S68A/S72A) down-regulated HIF2 comparably to wild-type protein, while pVHL30(S68D/S72D) did not (Fig. 9A; compare lanes 3 and 4), implying that the latter mutant is defective in targeting HIF2 for degradation. Consistent with this finding, levels of Glut-1 protein, a known transcriptional target of HIF2, remained high in cells expressing the pVHL30(S68D/S72D) mutant (Fig. 9A, lane 4). Cells expressing pVHL30(S65D) or pVHL30(S80D) regulated HIF2 levels like wild-type protein (data not shown).
Since pVHL30(S68D/S72D) failed to down-regulate HIF2 levels, we asked whether it is defective in pVHL/elongin C/elongin B (VCB)-cullin 2 (Cul2) E3 ligase complex formation. We subjected whole-cell extracts of the various cell lines to immunoprecipitation experiments using an anti-pVHL antibody (Fig. 9B). Anti-pVHL immunoprecipitates from 786-O vector cells failed to coimmunoprecipitate Cul2, a well-established E3 ligase partner protein of pVHL, while all pVHL species tested were able to interact with Cul2 comparably to wild-type protein (Fig. 9B). This suggests that E3 ligase complex formation is not altered in the pVHL30(S68D/S72D) mutant.
Next we tested whether the interaction of pVHL30(S68D/S72D) with HIF2 is affected. Thus, we analyzed the interaction of recombinant pVHL30(S68D/S72D) and HIF2 in vitro (Fig. 9C). In our experimental settings, wild-type pVHL30 bound HIF2 quite efficiently (about 20 to 30% of total input [data not shown]), while the binding of pVHL to HIF2 was reduced when pVHL that had previously been phosphorylated by recombinant GSK3 was used (Fig. 9C; compare lanes 1 and 2). Consistent with this observation, the pVHL30(S68D/S72D) mutant was completely defective in HIF2 binding (Fig. 9C, lane 3). These data would suggest that phospho-like modifications of pVHL on S68 and S72 negatively affect the direct interaction of pVHL with HIF2.
An analysis of the crystal structures of the VCB complex either unbound (38) or bound to a 20-amino-acid HIF1 peptide (15, 32) fully supports our biochemical observation (see Fig. S3 in the supplemental material). Overall, the structure of pVHL does not appear to be affected by HIF1 binding (see Fig. S3A and B in the supplemental material), but a closer look at S68 of pVHL revealed a striking difference. The carbonyl group of S68 serves as hydrogen-bonding partner for a key water molecule, thereby blocking the interaction of Arg69 (R69) with this water molecule (see Fig. S3C in the supplemental material). Upon HIF1 binding, this carbonyl group turns away from the water molecule, allowing R69 to act as a hydrogen bond acceptor for the water molecule (see Fig. S3D in the supplemental material). Furthermore, S72 of pVHL stabilizes S111 by two hydrogen bonds, thereby supporting the direct interaction of S111 with the hydroxyproline of HIF1 (see Fig. S3E in the supplemental material). Therefore, the existing structural data on the pVHL-HIF1 interaction also suggest that S68 and S72 of pVHL could contribute to the interaction of pVHL with HIF1 that is hydroxylated on proline 564.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MT binding and stabilization are properties of the VHL tumor suppressor protein that have been directly connected to the long and not the short form of pVHL (14). Notably, it is also the long form of pVHL that serves as an efficient substrate for GSK3 in vivo (Fig. 3D). In this regard, we have previously shown that pVHL30 localizes predominantly to the cytoplasm whereas pVHL19 is mostly nuclear (14). Thus, differential localization of these isoforms may contribute, at least in part, to the apparent selectivity of GSK3 for pVHL30.
Phosphorylation of pVHL30 by GSK3 influences pVHL30's MT stabilization and not MT binding activity (see Fig. 4). One possible outcome of these phosphorylation events might be recruitment/displacement of specific proteins to/from MT-bound pVHL30. In addition, phosphorylation of pVHL30 by GSK3 could regulate aspects of MT dynamics at specific subcellular sites, thereby contributing to the proper control of cellular processes such as cell polarization. Of particular interest in this regard is the fact that the MT-stabilizing activity of pVHL is restricted to sites in the cell periphery (29). Therefore, local inactivation of GSK3 could lead to a spatially restricted MT-stabilizing activity of pVHL in a fashion similar to that previously reported for the interplay of APC and GSK3 (10). Specifically, Hall and colleagues (10) have shown that Cdc42-mediated activation of PKC leads to inactivation of GSK3ß, followed by the association of APC with MT ends at the leading edge and stabilization of selected MTs. Considering these findings, further work on the factors that associate with pVHL30 in the performance of its MT-stabilizing function and its role at the cell periphery is clearly warranted.
Strikingly, phosphomimicking mutations on S68 and S72 affected not only the MT stabilization activity of pVHL but also the interaction of pVHL with HIF (Fig. 9). Taking this finding at face value, one is tempted to argue that the dual-kinase regulatory mechanism described here adds a new level to the regulation of the HIF pathway through negatively affecting pVHL-HIF interactions. An intriguing alternative interpretation is that phosphorylation of pVHL30 on S68 and S72 contributes to proper partitioning of total cellular pVHL into distinct pools that participate in the HIF pathway and MT dynamic control, respectively. However, further work will be required to distinguish between these possibilities.
The findings that a dual-kinase mechanism is involved in the regulation of pVHL's MT function and that at least one upstream kinase, GSK3, is part of signaling networks that play an important role in various aspects of tumor development (22, 33) raise the intriguing possibility that inactivation of this specific function of pVHL could be achieved through mechanisms other than genetic alterations of the VHL locus itself. In this regard, it is interesting that the incidence of retinal angioma cannot be correlated with specific germ line mutations in patients suffering from VHL disease (40). In fact, Maher and colleagues speculated that other genetic factors (and not VHL itself), may contribute to disease development, although such factors have not been identified as yet. Therefore, altered GSK3 signaling might represent such an alternative mechanism for pVHL tumor suppressor inactivation at the posttranslational level.
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ACKNOWLEDGMENTS |
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(wild-type and kinase-dead) constructs, and S. W. Lowe for the pLMP retrovirus vector. We are thankful to M. Cabrita and I. Frew for critical comments on the manuscript. This work was supported by the Steiner Foundation, the Novartis Research Foundation, and a grant from the Swiss National Science Foundation to W.K.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Present address: Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.
¶ J.L. and C.R.T. contributed equally to this work.
|| Present address: UCD School of Medicine and Medical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
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REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Barry, R. E., and W. Krek. 2004. The von Hippel-Lindau tumour suppressor: a multi-faceted inhibitor of tumourigenesis. Trends Mol. Med. 10:466-472.[CrossRef][Medline]
3. Beals, C. R., C. M. Sheridan, C. W. Turck, P. Gardner, and G. R. Crabtree. 1997. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275:1930-1934.
4. Blankenship, C., J. G. Naglich, J. M. Whaley, B. Seizinger, and N. Kley. 1999. Alternate choice of initiation codon produces a biologically active product of the von Hippel Lindau gene with tumor suppressor activity. Oncogene 18:1529-1535.[CrossRef][Medline]
5. Ciani, L., O. Krylova, M. J. Smalley, T. C. Dale, and P. C. Salinas. 2004. A divergent canonical WNT-signaling pathway regulates microtubule dynamics: dishevelled signals locally to stabilize microtubules. J. Cell Biol. 164:243-253.
6. Cohen, P., and S. Frame. 2001. The renaissance of GSK3. Nat. Rev. Mol. Cell Biol. 2:769-776.[CrossRef][Medline]
7. Dickins, R. A., M. T. Hemann, J. T. Zilfou, D. R. Simpson, I. Ibarra, G. J. Hannon, and S. W. Lowe. 2005. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37:1289-1295.[Medline]
8. Doble, B. W., and J. R. Woodgett. 2003. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 116:1175-1186.
9. Epstein, A. C., J. M. Gleadle, L. A. McNeill, K. S. Hewitson, J. O'Rourke, D. R. Mole, M. Mukherji, E. Metzen, M. I. Wilson, A. Dhanda, Y. M. Tian, N. Masson, D. L. Hamilton, P. Jaakkola, R. Barstead, J. Hodgkin, P. H. Maxwell, C. W. Pugh, C. J. Schofield, and P. J. Ratcliffe. 2001. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107:43-54.[CrossRef][Medline]
10. Etienne-Manneville, S., and A. Hall. 2003. Cdc42 regulates GSK-3ß and adenomatous polyposis coli to control cell polarity. Nature 421:753-756.[CrossRef][Medline]
11. Frame, S., P. Cohen, and R. M. Biondi. 2001. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol. Cell 7:1321-1327.[CrossRef][Medline]
12. Goold, R. G., and P. R. Gordon-Weeks. 2001. Microtubule-associated protein 1B phosphorylation by glycogen synthase kinase 3ß is induced during PC12 cell differentiation. J. Cell Sci. 114:4273-4284.
13. Goold, R. G., R. Owen, and P. R. Gordon-Weeks. 1999. Glycogen synthase kinase 3ß phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones. J. Cell Sci. 112:3373-3384.[Abstract]
14. Hergovich, A., J. Lisztwan, R. Barry, P. Ballschmieter, and W. Krek. 2003. Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL. Nat. Cell Biol. 5:64-70.[CrossRef][Medline]
15. Hon, W. C., M. I. Wilson, K. Harlos, T. D. Claridge, C. J. Schofield, C. W. Pugh, P. H. Maxwell, P. J. Ratcliffe, D. I. Stuart, and E. Y. Jones. 2002. Structural basis for the recognition of hydroxyproline in HIF-1 by pVHL. Nature 417:975-978.[CrossRef][Medline]
16. Iliopoulos, O., A. P. Levy, C. Jiang, W. G. Kaelin, Jr., and M. A. Goldberg. 1996. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc. Natl. Acad. Sci. USA 93:10595-10599.
17. Iliopoulos, O., M. Ohh, and W. G. Kaelin, Jr. 1998. pVHL19 is a biologically active product of the von Hippel-Lindau gene arising from internal translation initiation. Proc. Natl. Acad. Sci. USA 95:11661-11666.
18. Ivan, M., K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J. M. Asara, W. S. Lane, and W. G. Kaelin, Jr. 2001. HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292:464-468.
19. Jaakkola, P., D. R. Mole, Y. M. Tian, M. I. Wilson, J. Gielbert, S. J. Gaskell, A. Kriegsheim, H. F. Hebestreit, M. Mukherji, C. J. Schofield, P. H. Maxwell, C. W. Pugh, and P. J. Ratcliffe. 2001. Targeting of HIF- to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468-472.
20. Jia, J., K. Amanai, G. Wang, J. Tang, B. Wang, and J. Jiang. 2002. Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 416:548-552.[CrossRef][Medline]
21. Kaelin, W. G., Jr. 2002. Molecular basis of the VHL hereditary cancer syndrome. Nat. Rev. Cancer 2:673-682.[CrossRef][Medline]
22. Kim, L., and A. R. Kimmel. 2000. GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr. Opin. Genet. Dev. 10:508-514.[CrossRef][Medline]
23. Klein, P. S., and D. A. Melton. 1996. A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93:8455-8459.
24. Krylova, O., M. J. Messenger, and P. C. Salinas. 2000. Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3ß. J. Cell Biol. 151:83-94.
25. Lisztwan, J., G. Imbert, C. Wirbelauer, M. Gstaiger, and W. Krek. 1999. The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity. Genes Dev. 13:1822-1833.
26. Lisztwan, J., A. Marti, H. Sutterluty, M. Gstaiger, C. Wirbelauer, and W. Krek. 1998. Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway. EMBO J. 17:368-383.[CrossRef][Medline]
27. Liu, C., Y. Li, M. Semenov, C. Han, G. H. Baeg, Y. Tan, Z. Zhang, X. Lin, and X. He. 2002. Control of ß-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837-847.[CrossRef][Medline]
28. Lolkema, M. P., M. L. Gervais, C. M. Snijckers, R. P. Hill, R. H. Giles, E. E. Voest, and M. Ohh. 2005. Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain. J. Biol. Chem. 280:22205-22211.
29. Lolkema, M. P., N. Mehra, A. S. Jorna, M. van Beest, R. H. Giles, and E. E. Voest. 2004. The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery. Exp. Cell Res. 301:139-146.[CrossRef][Medline]
30. Lucas, F. R., R. G. Goold, P. R. Gordon-Weeks, and P. C. Salinas. 1998. Inhibition of GSK-3ß leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. J. Cell Sci. 111:1351-1361.[Abstract]
31. Maxwell, P. H., M. S. Wiesener, G. W. Chang, S. C. Clifford, E. C. Vaux, M. E. Cockman, C. C. Wykoff, C. W. Pugh, E. R. Maher, and P. J. Ratcliffe. 1999. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271-275.[CrossRef][Medline]
32. Min, J. H., H. Yang, M. Ivan, F. Gertler, W. G. Kaelin, Jr., and N. P. Pavletich. 2002. Structure of an HIF-1-pVHL complex: hydroxyproline recognition in signaling. Science 296:1886-1889.
33. Polakis, P. 2000. Wnt signaling and cancer. Genes Dev. 14:1837-1851.
34. Price, M. A., and D. Kalderon. 2002. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by glycogen synthase kinase 3 and casein kinase 1. Cell 108:823-835.[CrossRef][Medline]
35. Sanchez, C., M. Perez, and J. Avila. 2000. GSK3ß-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling. Eur. J. Cell Biol. 79:252-260.[CrossRef][Medline]
36. Schoenfeld, A., E. J. Davidowitz, and R. D. Burk. 1998. A second major native von Hippel-Lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor. Proc. Natl. Acad. Sci. USA 95:8817-8822.
37. Stambolic, V., L. Ruel, and J. R. Woodgett. 1996. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr. Biol. 6:1664-1668.[CrossRef][Medline]
38. Stebbins, C. E., W. G. Kaelin, Jr., and N. P. Pavletich. 1999. Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284:455-461.
39. Wagner, U., M. Utton, J. M. Gallo, and C. C. Miller. 1996. Cellular phosphorylation of tau by GSK-3ß influences tau binding to microtubules and microtubule organisation. J. Cell Sci. 109:1537-1543.[Abstract]
40. Webster, A. R., F. M. Richards, F. E. MacRonald, A. T. Moore, and E. R. Maher. 1998. An analysis of phenotypic variation in the familial cancer syndrome von Hippel-Lindau disease: evidence for modifier effects. Am. J. Hum. Genet. 63:1025-1035.[CrossRef][Medline]
41. Wirbelauer, C., H. Sutterluty, M. Blondel, M. Gstaiger, M. Peter, F. Reymond, and W. Krek. 2000. The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts. EMBO J. 19:5362-5375.[CrossRef][Medline]
42. Yanagawa, S., Y. Matsuda, J. S. Lee, H. Matsubayashi, S. Sese, T. Kadowaki, and A. Ishimoto. 2002. Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila. EMBO J. 21:1733-1742.[CrossRef][Medline]
43. Yoshimura, T., Y. Kawano, N. Arimura, S. Kawabata, A. Kikuchi, and K. Kaibuchi. 2005. GSK-3ß regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120:137-149.[CrossRef][Medline]
44. Zumbrunn, J., K. Kinoshita, A. A. Hyman, and I. S. Nathke. 2001. Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3ß phosphorylation. Curr. Biol. 11:44-49.[CrossRef][Medline]
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