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Originally published In Press as doi:10.1074/jbc.M710222200 on January 24, 2008

J. Biol. Chem., Vol. 283, Issue 13, 8678-8686, March 28, 2008
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Coordinated Activation of the Nuclear Ubiquitin Ligase Cul3-SPOP by the Generation of Phosphatidylinositol 5-Phosphate*

Matthew W. Bunce{ddagger}, Igor V. Boronenkov{ddagger}, and Richard A. Anderson§1

From the §Department of Pharmacology and the {ddagger}Biomolecular Chemistry Graduate Program, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, December 14, 2007 , and in revised form, January 22, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphoinositide signaling pathways regulate numerous processes in eukaryotic cells, including migration, proliferation, and survival. The regulatory lipid phosphatidylinositol 4,5-bisphosphate is synthesized by two distinct classes of phosphatidylinositol phosphate kinases (PIPKs), the type I and II PIPKs. Although numerous physiological functions have been identified for type I PIPKs, little is known about the functions and regulation of type II PIPK. Using a yeast two-hybrid screen, we identified an interaction between the type IIβ PIPK isoform (PIPKIIβ) and SPOP (speckle-type POZ domain protein), a nuclear speckle-associated protein that recruits substrates to Cul3-based ubiquitin ligases. PIPKIIβ and SPOP interact and co-localize at nuclear speckles in mammalian cells, and SPOP mediates the ubiquitylation of PIPKIIβ by Cul3-based ubiquitin ligases. Additionally, stimulation of the p38 MAPK pathway enhances the ubiquitin ligase activity of Cul3-SPOP toward multiple substrate proteins. Finally, a kinase-dead PIPKIIβ mutant enhanced ubiquitylation of Cul3-SPOP substrates. The kinase-dead PIPKIIβ mutant increases the cellular content of its substrate lipid phosphatidylinositol 5-phosphate (PI5P), suggesting that PI5P may stimulate Cul3-SPOP activity through a p38-dependent signaling pathway. Expression of phosphatidylinositol-4,5-bisphosphate 4-phosphatases that generate PI5P dramatically stimulated Cul3-SPOP activity and was blocked by the p38 inhibitor SB203580. Taken together, these data define a novel mechanism whereby the phosphoinositide PI5P leads to stimulation of Cul3-SPOP ubiquitin ligase activity and also implicate PIPKIIβ as a key regulator of this signaling pathway through its association with the Cul3-SPOP complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphoinositide signaling pathways modulate a diverse array of cellular processes in eukaryotes. Modification of the inositol ring by lipid kinases and phosphatases produces distinct phosphatidylinositol phosphate (PIP)2 isomers. These phosphatidylinositol phosphate isomers in turn selectively modulate the activities of effector proteins. In the cytosol, phosphoinositides regulate numerous processes, including actin polymerization, focal adhesion dynamics, ion channel activity, growth factor receptor signaling, and vesicle trafficking (1-4). In the nucleus, an autonomous phosphoinositide cycle regulates processes, including differentiation, proliferation, cell cycle progression, and apoptosis (5).

Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) is a critical phosphoinositide in eukaryotic cells. PI-4,5-P2 is not only itself a potent signaling molecule but is also a precursor of other second messengers such as phosphatidylinositol 3,4,5-trisphosphate, inositol 1,4,5-trisphosphate, and diacylglycerol. Because of its multipotent signaling capacity, the regulated synthesis of PI-4,5-P2 is essential for eukaryotes. In mammalian cells, PI-4,5-P2 is synthesized by two classes of phosphatidylinositol phosphate kinases (PIPKs) (6). Type I PIPKs (PIPKIs) preferentially use phosphatidylinositol 4-phosphate as a substrate to synthesize PI-4,5-P2, whereas type II PIPKs (PIPKIIs) generate PI-4,5-P2 from PI5P in a catalytically distinct reaction. Type I PIPKs modulate numerous cellular processes, including motility, focal adhesion assembly/disassembly, and vesicular trafficking (1-4). In contrast to type I PIPKs, little is known about the physiological functions of type II PIPKs. Although both PIPK subfamilies synthesize PI-4,5-P2, PIPKIIs are functionally non-redundant with PIPKIs (7-9). Thus PIPKIIs likely modulate a set of cellular processes distinct from those modulated by PIP-KIs. We have previously shown that PIPKIIβ is present within the nucleus at nuclear speckles, suggesting a role for PIPKIIβ in nuclear signaling pathways (10). Consistent with this hypothesis, PIPKIIβ and its substrate PI5P have recently been implicated within cellular stress-response pathways in the nucleus (11, 12).

The ubiquitylation and degradation of proteins is essential both for constitutive protein turnover as well as for the modulation of signaling pathways in response to extracellular stimuli. Ubiquitylation involves a three-step process in which ubiquitin is primed by an activating enzyme (E1), transferred to a conjugating enzyme (E2), and finally attached to a designated protein substrate by a ubiquitin ligase (E3). Specificity is mediated primarily by the multiprotein E3 ubiquitin ligases, which recruit unique substrates through a number of modular specificity factors. One of the best known E3 ubiquitin ligases in eukaryotes is the SCF (Skp1/Cul1/F-box) complex, a modular ubiquitin ligase assembled on the cullin protein Cul1. In addition to Cul1, six other cullins (Cul2, -3, -4A, -4B, 5, and -7) have been identified in humans. Cul3-based ubiquitin ligases are an emerging member of this family (13-16). Substrate specificity of Cul3-based ubiquitin ligases is dictated by BTB (Broad complex/Tramtrack/bric-a-brac) domain-containing proteins that bind directly to Cul3 through their BTB domain and bind substrates through a second protein-protein interaction domain (13, 14, 16, 17). Orthologs of Cul3 and BTB proteins have been identified in eukaryotes ranging from Caenorhabditis elegans to humans, and several substrates of Cul3-based ligases have been identified (18-20).

A central theme in the regulation of phosphoinositide signaling pathways is the interaction of enzymes such as PIPKs with upstream regulators and downstream effectors at discrete subcellular sites. To better understand the function and regulation of nuclear phosphoinositide signaling pathways, we sought to identify proteins that interact with PIPKIIβ. Yeast two-hybrid screening identified an interaction between PIPKIIβ and speckle-type POZ domain protein (SPOP), a nuclear speckle-associated BTB domain protein, and substrate adaptor for Cul3-based ubiquitin ligases (13, 14, 16, 17, 19). We demonstrate that PIPKIIβ and SPOP interact in vitro and in vivo and co-localize at nuclear speckles in HeLa cells. We also demonstrate that Cul3-SPOP mediates the ubiquitylation of PIPKIIβ in vivo, and that the ubiquitylation of multiple Cul3-SPOP substrates is potently stimulated by the MKK6-p38 MAPK pathway. Finally, we demonstrate that PI5P, the product of the PI-4,5-P2 4-phosphatases and the lipid substrate of PIPKIIβ, stimulates Cul3-SPOP activity, and this was blocked by the p38 inhibitor SB203580. Taken together, our data support a novel signaling pathway in which PI5P and PIPKIIβ regulate Cul3-SPOP ubiquitin ligase activity through p38 MAPK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Antibodies—HEK293 and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under a humidified atmosphere with 5% CO2. MG132 (Boston Biochem, Boston), SB203580, and SP600125 (EMD Bioscience, San Diego) were dissolved in Me2SO and added to the media where indicated. {alpha}-GST and {alpha}-T7 antibodies were purchased from EMD Bioscience. {alpha}-Myc and {alpha}-hemagglutinin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). {alpha}-FLAG antibody was purchased from Sigma.

cDNA Synthesis and Cloning—Total cellular RNA for cDNA synthesis was prepared from HEK293 cells using the RNeasy mini kit (Qiagen) following the manufacturer's instructions. cDNAs for SPOP, Cul3, and MKK6 were generated from total cellular RNA using the Qiagen One-step reverse transcription-PCR kit. Sequenced cDNAs were cloned into bacterial and mammalian expression vectors as described in the text. cDNA expression constructs for the mammalian PI-4,5-P2 4-phosphatases were a generous gift from Dr. Philip Majerus (Washington University, St. Louis).

Yeast Two-hybrid Screen—The PIPKIIβ cDNA was submitted to the University of Wisconsin Molecular Interaction Facility for two-hybrid screening. The kinase insert domain of PIP-KIIβ (Asp287-Met365) was cloned into a GAL4 fusion vector and screened against human five cDNA libraries according to Molecular Interaction Facility protocols.

Expression and Purification Recombinant Proteins—For expression of recombinant proteins, bacterial expression constructs were transformed into BL21(DE3)-competent cells (Novagen). Liquid cultures were inoculated with a single colony, and recombinant protein expression was induced by the addition of 1 mM isopropyl 1-thio-β-D-galactopyranoside. Following expression, bacteria were lysed by sonication in PBS containing 0.5% Triton X-100 and Complete mini protease inhibitor mixture (Roche Applied Science). His6 and GST fusion proteins were purified on HisTrap HP and GSTrap FF affinity columns, respectively (Amersham Biosciences), according to the manufacturer's instructions.

GST Pulldown and Co-immunoprecipitation Assays—For GST pulldown assays, equimolar amounts of purified recombinant protein were incubated in 50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, pH 8 in the presence of glutathione-Sepharose resin (Amersham Biosciences). After 4 h at 4 °C, the resin was pelleted at low speed and washed three times in reaction buffer. Bound protein was eluted from the resin by the addition of 2x Laemmli sample buffer, resolved by SDS-PAGE, and detected by Western blot. For co-immunoprecipitation assays, HEK293 cells were transfected via calcium phosphate precipitation by standard methods. 24 h after transfection, cells were washed in PBS and lysed by low amplitude sonication in 50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 0.1% deoxycholate, 0.5 mM EDTA, pH 8. Cell lysates were centrifuged for 15 min at 20,000 x g, and supernatant was recovered. Immunoprecipitations were performed by adding 3 µg of antibody and 20 µl of protein G-Sepharose resin per ml of cell lysate. After incubation at 25 °C for 3 h, the resin was pelleted and washed three times in lysis buffer. Bound protein was eluted in 2x Laemmli sample buffer, resolved by SDS-PAGE, and detected by Western blot.

Immunofluorescence—HeLa cells were seeded on glass coverslips and transfected using FuGENE 6 transfection reagent (Roche Applied Science). Twenty four hours after transfection, cells were washed in cold PBS and fixed in methanol. Primary antibodies were diluted to 1 µg/ml in PBS + 0.1% Triton X-100 + 3% bovine serum albumin and incubated on the coverslips at 37 °C. Coverslips were washed three times with PBST and incubated with fluorophore-conjugated secondary antibodies for 1 h at 37 °C. After being washed with PBST, coverslips were mounted onto microscope slides using VectaShield mounting medium (Vector Laboratories). Visualization was performed using a Bio-Rad MRC-1024 laser scanning confocal microscope (W. M. Keck Laboratory for Biological Imaging, Madison, WI).

In Vivo Ubiquitylation Assays—HEK293 cells were transiently transfected with the indicated combinations of expression vectors by calcium phosphate precipitation. 24 h after transfection, MG132 was added to the media at a final concentration of 10 µM, and cells were incubated for 6 h at 37 °C. Cells were washed twice with PBS, lysed in PBS with 8 M urea and 0.2% SDS, and sonicated to reduce viscosity. Lysates were incubated for 3 h at 25 °C with nickel-Sepharose resin (Amersham Biosciences). After washing the resin three times with lysis buffer, proteins were eluted in sample buffer, resolved via SDS-PAGE, and detected by Western blot.


Figure 1
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  		FIGURE 1.
The kinase insert domain of PIPKIIβ mediates nuclear targeting. A, HeLa cells seeded on glass coverslips were transiently transfected with wild type PIPKIIβ or a PIPKIIβ({Delta}Ins) deletion mutant. After 24 h, cells were fixed, and PIPKIIβ was visualized by indirect immunofluorescence. B, HeLa cells were transfected with wild type β-galactosidase expression vector or a PIPKIIβ kinase insert/β-gal fusion vector (Ins-LacZ). Cells were analyzed as in A.

 


Figure 2
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  		FIGURE 2.
PIPKIIβ interacts with the nuclear speckle-associated protein SPOP. A, schematic illustrations of PIPKIIβ and SPOP. The kinase insert domain of PIPKIIβ used as bait and the SPOP clone identified in the two-hybrid screen are indicated. B, purified recombinant PIPKIIβ was incubated with GST, GST-SPOP, GST-MATH, or GST-BTB, and binding was analyzed by GST pulldown assays. C, purified recombinant PIPKIIβ or PIPKII{alpha} was incubated with GST or GST-SPOP, and binding was analyzed by GST pulldown assays.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Kinase Insert Domain of PIPKIIβ Is Necessary and Sufficient for Nuclear Targeting—We have demonstrated previously the presence of the type I{alpha} and type IIβ PIPK isoforms within mammalian nuclei, including their targeting to nuclear speckles (10). To identify the domain within PIPKIIβ required for its nuclear targeting, a panel of deletion mutants was generated, and their subcellular distributions were assessed by indirect immunofluorescence microscopy. The kinase insert domain of PIPKIIβ was required for nuclear localization, as its deletion prevented nuclear targeting of PIPKIIβ (Fig. 1A). These results are consistent with previous reports that an acidic {alpha}-helix within the kinase insert domain promotes nuclear targeting of PIPKIIβ (36, 37). To determine whether the PIPKIIβ kinase insert domain is sufficient for PIPKIIβ nuclear targeting, the kinase insert was fused to LacZ, and its subcellular localization was examined. Whereas a LacZ control was localized entirely within the cytosol, the kinase insert fusion (Ins-LacZ) targeted to the nucleus (Fig. 1B). These results identified the kinase insert domain of PIPKIIβ as being both necessary and sufficient for its nuclear targeting.

Identification of SPOP as a PIPKIIβ-binding Protein—Although the PIPKIIβ kinase insert mediates nuclear translocation, it does not contain a canonical nuclear localization signal. We therefore hypothesized that PIPKIIβ is targeted to the nucleus through its association with interacting partners. To identify proteins that interact with PIPKIIβ, we performed a yeast two-hybrid screen against several human cDNA libraries using the PIPKIIβ kinase insert domain as bait (Fig. 2A). One of the proteins identified by the two-hybrid screen was SPOP (speckle-type POZ domain protein) (Fig. 2A), a nuclear speckle-associated protein and a substrate specificity factor for Cul3-based ubiquitin ligases (18-20).

SPOP Interacts Specifically with PIPKIIβ in Vitro and in Vivo—The interaction between PIPKIIβ and SPOP was assessed using both in vitro and in vivo binding assays. To assess their in vitro association, GST pulldown assays were performed with recombinant purified GST-SPOP and His6-PIPKIIβ. PIPKIIβ was specifically retained by GST-SPOP but not by GST alone (Fig. 2B), confirming their interaction. SPOP contains two conserved domains: an N-terminal MATH (Meprin and Traf Homology) domain and a C-terminal BTB domain. To assess the contribution of each domain to the interaction between SPOP and PIPKIIβ, GST pulldown assays were performed with purified recombinant GST-MATH and GST-BTB proteins. PIPKIIβ co-precipitated with GST-MATH but not GST-BTB (Fig. 2B), demonstrating that SPOP interacts with PIPKIIβ through its MATH domain. To assess the specificity of SPOP for PIPKIIβ, the highly similar PIPKII{alpha} isoform was also tested. Despite ~80% primary sequence identity between the two PIPKII isoforms, SPOP did not interact with PIPKII{alpha} (Fig. 2C).

The interaction between PIPKIIβ and SPOP was also tested in vivo. HEK293 cells were transiently transfected with mammalian expression constructs of PIPKIIβ and SPOP. Twenty four hours after transfection, PIPKIIβ and SPOP were immunoprecipitated from cell lysates, resolved by SDS-PAGE, and probed by Western blot. PIPKIIβ and SPOP co-precipitated with each other, confirming their in vivo interaction (Fig. 3). Similar to in vitro results, PIPKII{alpha} did not co-precipitate with SPOP. The interaction of endogenous PIPKIIβ and SPOP was also assessed. Endogenous PIPKIIβ was immunoprecipitated from HEK293 lysates using either an {alpha}-PIPKIIβ antibody or normal rabbit IgG. Western blot analysis identified endogenous SPOP in the PIPKIIβ immunoprecipitates, thus confirming the interaction of endogenous PIP-KIIβ and SPOP proteins (Fig. 3C).

PIPKIIβ Co-localizes with SPOP at Nuclear Speckles—Having confirmed their interaction, we next assessed whether PIP-KIIβ and SPOP co-localize within the nucleus. HeLa cells were transiently transfected with SPOP and PIPKIIβ, and their subcellular distributions were analyzed by indirect immunofluorescence microscopy. A distinct pool of PIPKIIβ co-localized with SPOP at nuclear speckles (Fig. 4). In contrast, PIPKII{alpha} was present exclusively within the cytosol.


Figure 3
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  		FIGURE 3.
PIPKIIβ and SPOP interact in vivo. A, HEK293 cells were transiently transfected with HA-SPOP and either myc-PIPKIIβ or myc-PIPKII{alpha}. Twenty four hours after transfection, cells were lysed and immunoprecipitated (IP) with epitope tag-specific antibodies. Normal IgG was used as a negative control. B, endogenous PIPKIIβ was immunoprecipitated from HEK293 cell lysates using an {alpha}-PIPKIIβ antibody or normal rabbit IgG. Immunoprecipitates were resolved by SDS-PAGE and probed with {alpha}-PIPKIIβ or {alpha}-SPOP antibodies. HA, hemagglutinin.

 


Figure 4
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  		FIGURE 4.
PIPKIIβ co-localizes with SPOP at nuclear speckles. HeLa cells were transiently transfected with HA-SPOP and the indicated Myc-tagged PIPKII constructs. Twenty four hours after transfection, cells were fixed with methanol, and the transfected proteins were visualized by indirect immunofluorescence microscopy using epitope tag-specific antibodies.

 
PIPKIIβ and PIPKII{alpha} share ~80% primary sequence identity, with the greatest regions of diversity at the N and C termini and within the kinase insert domain. The specific interaction and co-localization of SPOP with PIPKIIβ therefore suggested that the PIPKIIβ kinase insert is necessary for its co-localization with SPOP at nuclear speckles. To test this hypothesis, chimeric PIPKII mutants were generated in which the kinase insert domains of PIPKII{alpha} and PIPKIIβ were exchanged, and their subcellular distributions were analyzed. PIPKII{alpha}(IIβ KI), which contains the PIPKIIβ kinase insert, co-localized with SPOP at nuclear speckles similar to wild type PIPKIIβ. Conversely, PIPKIIβ(II{alpha} KI), which contains the PIPKII{alpha} kinase insert, showed a cytosolic distribution similar to wild type PIPKII{alpha}. These results demonstrate that the PIPKIIβ kinase insert domain mediates its co-localization with SPOP at nuclear speckles.

SPOP Promotes the Ubiquitylation of PIPKIIβ by Cul3-based Ubiquitin Ligases—The Cul3-SPOP ubiquitin ligase complex has been shown to mediate the ubiquitylation and subsequent degradation of Daxx (19). We therefore assessed whether Cul3-SPOP also promotes the ubiquitylation and degradation of PIP-KIIβ. To assess PIPKIIβ turnover, HEK293 cells were transiently transfected with Cul3, SPOP, and the RING-box protein Rbx1, which is required for the activation of cullin-based ligases (21). Endogenous PIPKIIβ levels were assessed by Western blot either 24 or 48 h after transfection. Interestingly, no discernible change in endogenous PIPKIIβ protein levels was detected in transfected cells (data not shown), suggesting that the Cul3-SPOP ubiquitin ligase may not promote PIPKIIβ turnover. However, as only a fraction of PIPKIIβ (~20%) is nuclear, it would be difficult to detect enhanced turnover of nuclear PIP-KIIβ in the context of the total cellular PIPKIIβ.

Next, the ability of Cul3-SPOP to ubiquitylate PIPKIIβ was tested. PIPKIIβ was co-expressed in HEK293 cells with Cul3, Rbx1, SPOP, and His6-ubiquitin. After treating cells with the proteasome inhibitor MG132, cell lysates were purified over nickel resin to capture ubiquitylated proteins (22). Co-expression of the Cul3-SPOP complex promoted ubiquitylation of PIPKIIβ, detected as a ladder of high molecular weight protein by Western blot (Fig. 5A). Efficient ubiquitylation of PIPKIIβ required the Cul3-SPOP complex, as excluding either SPOP or Cul3 and Rbx1 failed to generate ubiquitylated PIPKIIβ. To assess the specificity of the Cul3-SPOP ligase complex for PIPKIIβ, ubiquitylation of PIPKIIβ and PIPKII{alpha} was compared. In contrast to PIPKIIβ, no ubiquitylation of PIPKII{alpha} was observed (Fig. 5B), demonstrating that SPOP specifically promotes ubiquitylation of PIPKIIβ in vivo.

PIPKIIβ Ubiquitylation by Cul3-SPOP Is Stimulated by MKK6/p38 MAPK—p38 MAPK has been shown to phosphorylate PIPKIIβ in response to UV irradiation and oxidative stress, thereby repressing PIPKIIβ lipid kinase activity (12). We hypothesized that p38 might also modulate PIPKIIβ ubiquitylation. Consistent with this hypothesis, a constitutively active mutant of the p38-activating kinase MKK6 (MKK6+) dramatically enhanced PIPKIIβ ubiquitylation in HEK293 cells (Fig. 5C). The MKK6-dependent p38 MAPK activation was blocked by the p38 inhibitor SB203580, further supporting a role for p38 in PIPKIIβ ubiquitylation. Importantly, MKK6+ did not promote PIPKIIβ ubiquitylation in the absence of the Cul3-SPOP complex (Fig. 5C), confirming that Cul3-SPOP is required for p38-stimulated ubiquitylation.


Figure 5
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  		FIGURE 5.
SPOP mediates the ubiquitylation of PIPKIIβ by Cul3-based ubiquitin ligases. A, HEK293 cells were transfected with myc-PIPKIIβ, His6-ubiquitin, and the indicated combinations of FLAG-Cul3, HA-Rbx1, and HA-SPOP. Ub, ubiquitin; IB, immunoblot. 24 h after transfection cells were treated with 10 µM MG132 for 6 h and lysed in the presence of 8 M urea, and ubiquitylated proteins were purified over nickel-Sepharose resin. Ubiquitylated PIP-KIIβ was detected using {alpha}-Myc antibodies. B, HEK293 cells were transfected with the indicated expression constructs. myc-PIPKIIβ and myc-PIPKII{alpha} were analyzed for ubiquitylation as in A. C, HEK293 cells were transfected with the indicated combinations of myc-PIPKIIβ, FLAG-Cul3, HA-Rbx1, HA-SPOP, and HA-MKK6+. 24 h after transfection, cells were treated with MG132, and PIP-KIIβ ubiquitylation was detected as described above. D, HEK293 cells were transfected with the indicated combinations of FLAG-Cul3, HA-Rbx1, HA-SPOP, HA-MKK6+, and either myc-PIPKIIβ or myc-PIPKIIβ(S326A). 24 h after transfection, cells were treated with MG132, and PIPKIIβ ubiquitylation was detected as described above.

 
PIPKIIβ is phosphorylated at Ser326 by p38 MAPK, and phosphorylation is sufficient to inhibit PIPKIIβ lipid kinase activity in vitro (12). To determine whether phosphorylation of PIPKIIβ at Ser326 also modulates its ubiquitylation, the ability of MKK6+ to stimulate ubiquitylation of a PIPKIIβ(S326A) point mutant was assessed. Interestingly, PIPKIIβ(S326A) ubiquitylation was stimulated by MKK6+ similar to wild type PIPKIIβ (Fig. 5D). Thus p38 MAPK stimulates PIPKIIβ ubiquitylation by a mechanism independent of Ser326 phosphorylation.

PI5P Activates Cul3-SPOP Ubiquitin Ligase Activity toward Multiple Substrates through a Pathway Inhibited by SB203580—Because PIPKIIβ associates with Cul3-SPOP, we hypothesized that PIPKIIβ ubiquitylation might be modulated by changes in local concentrations of either PI5P or PI-4,5-P2. Previous studies demonstrate that inhibiting PIPKIIβ, for example by p38-dependent attenuation of PIPKIIβ activity or RNA interference-mediated knockdown of endogenous PIPKIIβ, or expression of the kinase-dead PIPKIIβ, causes an increase in cellular PI5P levels (11, 12). To test the effect of elevated PI5P levels on PIPKIIβ ubiquitylation, the ubiquitylation of wild type PIPKIIβ and a well characterized kinase-dead PIPKIIβ point mutant, PIPKIIβ(D278A), were compared side-by-side. Ubiquitylation of PIPKIIβ(D278A) was dramatically enhanced when compared with the wild type protein (Fig. 6A); furthermore, inhibiting p38 MAPK with SB203580 reduced PIPKIIβ(D278A) ubiquitylation (Fig. 6B). These results suggested that increased cellular PI5P levels stimulate PIPKIIβ ubiquitylation by Cul3-SPOP, and that this stimulation is transduced by p38 MAPK.

As an independent method to test the hypothesis that PI5P generation stimulates the Cul3-SPOP ubiquitin ligase complex, the ubiquitylation of wild type PIPKIIβ by Cul3-SPOP was analyzed by co-expressing either of two recently characterized PI-4,5-P2 4-phosphatases (23). Both PI-4,5-P2 4-phosphatases caused a strong Cul3-SPOP-dependent increase in PIPKIIβ ubiquitylation, similar to the results seen with co-expression of MKK6+ (Fig. 6B). This effect was blocked by SB203580, illustrating a requirement for p38 MAPK downstream of PI5P. Importantly, neither MKK6+ nor the PI-4,5-P2 4-phosphatases caused a detectable change in total cellular ubiquitylation (data not shown), reinforcing the specificity of this pathway for ubiquitylation of Cul3-SPOP substrates.

Our observations that PI5P and p38 stimulate the ubiquitylation of PIPKIIβ suggested that these signals may cause a general enhancement of Cul3-SPOP activity, thereby promoting the ubiquitylation of numerous Cul3-SPOP substrates. To test this possibility, the effects of PI5P and p38 MAPK on the ubiquitylation of the Fas receptor binding protein Daxx and the pancreatic transcription factor Pdx1 were analyzed. Daxx is a confirmed Cul3-SPOP substrate (19, 24); Pdx1 has not previously been shown to be ubiquitylated by Cul3-SPOP but is a known SPOP-interacting protein, and its transcriptional activity is negatively regulated by SPOP (25, 26). As we observed with PIPKIIβ, both Daxx and Pdx1 were ubiquitylated in vivo by Cul3-SPOP, and their ubiquitylation was stimulated by MKK6+ in an SB203580-sensitive manner (Fig. 7A). Furthermore, co-expression of the type I PI-4,5-P2 4-phosphatase stimulated the ubiquitylation of both Daxx and Pdx1 (Fig. 7B). Ubiquitylation was attenuated by SB203580, but not by the JNK-specific inhibitor SP600125, reinforcing a specific role for p38 MAPK.

As a complementary approach, the ubiquitylation of Daxx was also assessed in cells overexpressing the kinase-dead PIPKIIβ(D278A) mutant. Kinase-dead PIPKIIβ enhanced the activity of the Cul3-SPOP complex toward Daxx as shown in Fig. 7C. This was also p38 MAPK-dependent, as SB203580 attenuated the effect.


Figure 6
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  		FIGURE 6.
PI5P enhances ubiquitylation of PIPKIIβ. A, HEK293 cells were transfected with FLAG-Cul3, HA-Rbx1, HA-SPOP, and either wild type (WT) or kinase-dead PIPKIIβ (PIPKIIβ(D278A)). 24 h after transfection, cells were treated with MG132 and lysed, and PIPKIIβ ubiquitylation was analyzed. IB, immunoblot. B, ubiquitylation of kinase-dead PIPKIIβ was analyzed as in A in the absence or presence of 2 µM SB203580. C, HEK293 cells were transfected with the indicated combinations of myc-PIPKIIβ, FLAG-Cul3, HA-Rbx1, HA-SPOP, and FLAG-tagged type I or type II PI-4,5-P2 4-phosphatase. PIPKIIβ ubiquitylation was analyzed 24 h after transfection.

 


Figure 7
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  		FIGURE 7.
PI5P stimulates Cul3-SPOP activity toward multiple substrates. A, myc-Daxx or myc-Pdx1 were expressed in HEK293 cells with the indicated combinations of FLAG-Cul3, HA-Rbx1, HA-SPOP, and HA-MKK6+. Ubiquitylation of Daxx and Cul3 was analyzed 24 h after transfection. IB, immunoblot. B, HEK293 cells were transfected with FLAG-Cul3, HA-Rbx1, HA-SPOP, FLAG-type 1 PI-4,5-P2 4-phosphatase, and either myc-Daxx or myc-Pdx1. Where indicated, transfected cells were treated with SB203580 or SP600125. Ubiquitylation of Daxx and Pdx1 was analyzed 24 h after transfection. C, HEK cells were transfected with the indicated combinations of FLAG-Cul3, HA-Rbx1, HA-SPOP, myc-Daxx, and either HA-MKK6+, FLAG-type I PI-4,5-P2 4-phosphatase, or kinase-dead FLAG-PIPKIIβ. Where indicated, cells were treated with SB203580. Ubiquitylation of Daxx was assessed 24 h after transfection.

 
SPOP Is Also Ubiquitylated Downstream of PI5P and MKK6—Several F-box and SOCS proteins have been shown previously to be degraded by Cul1 and Cul2 ubiquitin ligases, respectively (27-29). The C. elegans BTB protein MEL-26 also appears to be degraded by Cul3 ligases (14, 15), suggesting a general mechanism whereby substrate adaptor proteins like SPOP are degraded along with their recruited substrates. To determine whether SPOP is ubiquitylated in vivo, myc-SPOP was expressed in HEK293 cells with His6-Ub, and its ubiquitylation was assessed. Ubiquitylated myc-SPOP was readily detected in these assays (Fig. 8B), confirming its in vivo ubiquitylation.


Figure 8
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  		FIGURE 8.
SPOP is ubiquitylated by Cul3 downstream of p38 MAPK. A, diagrams of wild type (WT) SPOP and SPOP truncation mutants assayed for ubiquitylation. B, HEK293 cells were transiently transfected with His6-ubiquitin and the indicated SPOP expression vectors. 24 h after transfection, SPOP was analyzed for ubiquitylation as described above. IB, immunoblot. C, HEK293 cells were transfected with myc-SPOP and the indicated combinations of FLAG-Cul3, HA-Rbx1, and HA-MKK6+. SPOP ubiquitylation was analyzed 24 h after transfection.

 


Figure 9
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  		FIGURE 9.
Model of PI5P-and p38-sensitive regulation of Cul3-SPOP activity. Under resting conditions, PIPKIIβ maintains low PI5P levels by synthesizing PI-4,5-P2. In response to a stimulus such as UV irradiation or oxidative stress, PIPKIIβ activity is attenuated, resulting in accumulation of PI5P. PI5P modulates an upstream activator of p38 MAPK, resulting in the general activation of Cul3-SPOP toward multiple substrates. Ub, ubiquitin.

 
SPOP recruits substrate proteins through its N-terminal MATH domain and interacts with Cul3 through its C-terminal BTB domain. To assess if SPOP is ubiquitylated specifically within either of these conserved domains, several SPOP truncation mutants lacking either their N- or C-terminal domains were constructed (Fig. 8A) and expressed in HEK293 cells. Deletion of the C-terminal domain of SPOP did not prevent its ubiquitylation (Fig. 8B). In contrast, deletion of the N-terminal domain of SPOP prevented ubiquitylation, demonstrating that SPOP is ubiquitylated specifically within its N-terminal domain.

Finally, we assessed the effects of Cul3/Rbx1 and p38 MAPK on SPOP ubiquitylation. Compared with expression of SPOP alone, expression of Cul3/Rbx1 caused an increase in SPOP ubiquitylation, whereas expression of MKK6+ had no effect in the absence of Cul3-SPOP (Fig. 8C). Similar to the results observed with PIPKIIβ, co-expression of Cul3, Rbx1, and MKK6+ caused a strong increase in SPOP ubiquitylation that was attenuated by SB203580. Similar results were observed when the PIPKIIβ, Daxx, and Pdx1 Western blots in Figs. 5, 6, and 7 were re-probed with {alpha}-SPOP antibodies (data not shown). Thus we conclude that SPOP is ubiquitylated along with its cargo in a pathway enhanced by PI5P and p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The regulated turnover of signaling proteins is critical for both the induction and attenuation of pathways crucial for cell growth, proliferation, and survival. Modular E3 ubiquitin ligases provide a mechanism by which specific proteins are recruited for ubiquitylation through their interaction with substrate-specific adaptor proteins. Here, we have demonstrated that the nuclear phosphatidylinositol phosphate kinase PIP-KIIβ interacts specifically and co-localizes with the Cul3 adaptor protein SPOP. The Cul3-SPOP ubiquitin ligase complex ubiquitylates PIPKIIβ in vivo, and ubiquitylation is potently stimulated by the MKK6/p38 MAPK pathway. Consistent with previous reports, SPOP itself is also ubiquitylated within the Cul3-SPOP complex. Most significantly, we have demonstrated that PI5P, the lipid substrate of PIPKIIβ, stimulates Cul3-SPOP activity toward multiple substrates, including the SPOP-binding proteins Daxx and Pdx1. The ability of the p38-specific inhibitor SB203580 to prevent Cul3-SPOP stimulation further suggests that PI5P functions by activating the p38 MAPK pathway. Together, these results define a novel mechanism whereby phosphoinositide signaling may modulate the functions of key signaling proteins by regulating the activity of a nuclear ubiquitin ligase. Based on our experimental data, we propose the model illustrated in Fig. 9.

p38 MAPK has been shown previously to directly inhibit the lipid kinase activity of PIPKIIβ through phosphorylation of Ser326. Here we have shown that p38 MAPK also stimulates PIPKIIβ ubiquitylation independently of Ser326 phosphorylation. Furthermore, we have shown that p38 stimulates the ubiquitylation of multiple Cul3-SPOP substrate proteins, including Daxx and Pdx1. These data demonstrate that p38 stimulates the Cul3-SPOP ubiquitin ligase activity, although currently the mechanism by which this occurs is unknown. One possibility is that either p38 or a p38 effector kinase phosphorylates Cul3-SPOP, thereby stimulating its catalytic activity. In support of this hypothesis, p38 has been reported to phosphorylate the RING E3 ubiquitin ligase Siah2, increasing its activity toward its substrate PHD3 (30). Further characterization is required to determine how p38 activation results in stimulation of Cul3-SPOP activity.

A key discovery from our experiments is the ability of PI5P to stimulate Cul3-SPOP activity through a p38-dependent signaling pathway. Although relatively little about the physiological functions of PIPKIIβ is currently known, one recurring theme is the role of its substrate, PI5P, as a key regulatory molecule. Previous reports have linked PIPKIIβ and PI5P to the modulation of insulin signaling and of a nuclear stress-response pathway (12, 31, 32). In both of these pathways, the primary function of PIPKIIβ appears to be regulating PI5P levels by converting it to PI-4,5-P2. These observations provide an intriguing contrast to the canonical Type I PIPK signaling pathways, in which PI-4,5-P2 is the key regulatory species modulating effector proteins. Our results now identify a third signaling pathway in which PI5P elicits a potent regulatory effect, and suggest that PIPKIIβ regulates the system by maintaining low PI5P levels under resting conditions. If PIPKIIβ is primarily a regulator of Cul3-SPOP rather than a substrate, this might explain our observation that endogenous PIPKIIβ is not degraded upon overexpression of Cul3-SPOP in cultured cells.

Although our data provide significant insight into a novel phosphoinositide-regulated signaling pathway, numerous questions remain. Physiological stimuli that activate the PI5P-sensitive stimulation of Cul3-SPOP have yet to be identified. Because activation of Cul3-SPOP is p38-sensitive, it is possible that Cul3-SPOP mediates a stress-response pathway. The previous characterization of PI5P as a transducer of a p38-dependent stress-response pathway supports this hypothesis (12). Additionally, a stress-response model would be consistent with the observation that Daxx, which modulates survival and apoptosis signaling pathways, is itself ubiquitylated by Cul3-SPOP. The mechanism by which PI5P activates p38 MAPK is also currently unknown, for example whether PI5P directly stimulates an upstream kinase of the p38 cascade. Additionally, the role of cytosolic versus nuclear PI5P in p38-dependent activation of Cul3-SPOP remains to be defined. Distinguishing between these two pools of PI5P will help to more thoroughly characterize the signaling pathway leading to activation of Cul3-SPOP. Finally, the physiological effects of Cul3-SPOP remain poorly understood, e.g. whether Cul3-SPOP is primarily involved with stress-response pathways or if it modulates numerous other pathways in the cell. This uncertainty is due in part to the lack of known Cul3-SPOP substrates. As new substrates continue to be identified and characterized, the principal physiological function(s) of Cul3-SPOP will become increasingly clear. Further investigation of these and other aspects will be critical for a more complete understanding of how phosphoinositide signaling contributes to the regulation of Cul3-SPOP and its substrates, and how PI5P, p38 MAPK, and Cul3-SPOP contribute to cell physiology.

Our observation that SPOP is ubiquitylated by the Cul3 ubiquitin ligase complex is consistent with previous reports that substrate adaptors for Cul1, Cul2, and Cul3 E3 ligases are ubiquitylated in an autocatalytic mechanism. Our data specifically identify the N-terminal half of SPOP containing the MATH domain as the major ubiquitylation target. Structural analyses of cullin E3 ligases indicate that cullin E3s form rigid scaffolds that orient E2-conjugating enzymes in close proximity to their substrates, and that the E2s only transiently associate with the E3 complex (33-35). Therefore, proper orientation is critical for efficient ubiquitylation of a substrate protein. Because BTB proteins such as SPOP interact with their substrates through their MATH domains, the MATH domain would likely be in close proximity to the E2-conjugating enzyme. In contrast, the BTB domain of SPOP, through which it interacts with Cul3, would be more distal to the E2 enzyme and therefore a less efficient target for ubiquitylation. Other cullin specificity factors may be similarly ubiquitylated within their substrate-binding domains. A more detailed analysis would be beneficial to more thoroughly understand the function of cullin specificity factors as well as the activities of cullin-based ubiquitin ligases as a whole.

In summary, the data presented in this study define a novel mechanism by which phosphoinositide signaling regulates a nuclear ubiquitin ligase complex. The interaction of PIPKIIβ with the Cul3-SPOP ubiquitin ligase, coupled with the ability of the PIPKIIβ substrate PI5P to modulate Cul3-SPOP activity through a signaling pathway inhibited by the p38 inhibitor SB203580, identifies a new function for phosphoinositide signaling within the nucleus. Identification of stimuli that enhance or attenuate this pathway, as well as delineation of the down-stream physiological effects, will be invaluable to more thoroughly understand the roles of phosphoinositides within the nucleus.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 1300 University Ave., Rm. 3750 MSC, Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}wisc.edu.

2 The abbreviations used are: PIP, phosphatidylinositol phosphate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PIPK, phosphatidylinositol phosphate kinase; PIPKI, PIPK type I; PIPKII, PIPK type II; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; BTB, Broad complex/Tramtrack/bric-a-brac. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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