Regulation of Postsynaptic RapGAP SPAR by Polo-like Kinase 2 and the SCFβ-TRCP Ubiquitin Ligase in Hippocampal Neurons
2008; Elsevier BV; Volume: 283; Issue: 43 Linguagem: Inglês
10.1074/jbc.m802475200
ISSN1083-351X
AutoresXiaolu L. Ang, Daniel P. Seeburg, Morgan Sheng, J. Wade Harper,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe ubiquitin-proteasome pathway (UPP) regulates synaptic function, but little is known about specific UPP targets and mechanisms in mammalian synapses. We report here that the SCFβ-TRCP complex, a multisubunit E3 ubiquitin ligase, targets the postsynaptic spine-associated Rap GTPase activating protein (SPAR) for degradation in neurons. SPAR degradation by SCFβ-TRCP depended on the activity-inducible protein kinase Polo-like kinase 2 (Plk2). In the presence of Plk2, SPAR physically associated with the SCFβ-TRCP complex through a canonical phosphodegron. In hippocampal neurons, disruption of the SCFβ-TRCP complex by overexpression of dominant interfering β-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR. Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function. The ubiquitin-proteasome pathway (UPP) regulates synaptic function, but little is known about specific UPP targets and mechanisms in mammalian synapses. We report here that the SCFβ-TRCP complex, a multisubunit E3 ubiquitin ligase, targets the postsynaptic spine-associated Rap GTPase activating protein (SPAR) for degradation in neurons. SPAR degradation by SCFβ-TRCP depended on the activity-inducible protein kinase Polo-like kinase 2 (Plk2). In the presence of Plk2, SPAR physically associated with the SCFβ-TRCP complex through a canonical phosphodegron. In hippocampal neurons, disruption of the SCFβ-TRCP complex by overexpression of dominant interfering β-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR. Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function. Dendritic spines are tiny, actin-rich, dynamic protrusions radiating from the dendritic shaft of principal neurons and comprise the postsynaptic compartment of most glutamatergic synapses of the mammalian brain. The size and morphology of dendritic spines are correlated with their function. Thus, large mushroom-shaped spines tend to be more stable than small thin spines, contain more α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors, and mediate stronger synaptic connections (1Kasai H. Matsuzaki M. Noguchi J. Yasumatsu N. Nakahara H. Trends Neurosci. 2003; 26: 360-368Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 2Tada T. Sheng M. Curr. Opin. Neurobiol. 2006; 16: 95-101Crossref PubMed Scopus (527) Google Scholar). The morphology of spines, which changes during development and in response to synaptic activity, is influenced by multiple signaling pathways that emanate from postsynaptic glutamate receptors and act upon the actin cytoskeleton and associated proteins in the postsynaptic density (PSD) 5The abbreviations used are: PSD, postsynaptic density; Ub, ubiquitin; GAP, GTPase activating protein; SPAR, spine-associated Rap GTPase activating protein; Plk2, Polo-like kinase 2; UPP, ubiquitin-proteasome pathway; CMV, cytomegalovirus; HA, hemagglutinin; GFP, green fluorescent protein; SCF, Skp1/Cul1/F-box protein; WT, wild type; RFP, red fluorescent protein; GST, glutathione S-transferase; RNAi, RNA-mediated interference; DIV, days in vitro; shRNA, short hairpin RNA; E3, ubiquitin-protein isopeptide ligase; β-TRCP, β-transducin repeat-containing protein. (2Tada T. Sheng M. Curr. Opin. Neurobiol. 2006; 16: 95-101Crossref PubMed Scopus (527) Google Scholar, 3Bonhoeffer T. Yuste R. Neuron. 2002; 35: 1019-1027Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 4Carlisle H.J. Kennedy M.B. Trends Neurosci. 2005; 28: 182-187Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). SPAR (spine-associated Rap GTPase activating protein (GAP)) is a PSD protein that regulates spine morphogenesis and forms a complex with the scaffold protein PSD-95 and N-methyl-d-aspartate-type glutamate receptors (5Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Overexpression of SPAR results in enlargement of spine heads, an effect dependent upon the ability of SPAR to rearrange actin and function as a RapGAP. In contrast, dominant-negative SPAR produces long and thin spines (5Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). SPAR, in turn, is regulated by polo-like kinase 2 (Plk2; also known as serum-inducible serine/threonine kinase (SNK)) (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). Synaptic activity induces Plk2 expression (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar, 7Kauselmann G. Weiler M. Wulff P. Jessberger S. Konietzko U. Scafidi J. Staubli U. Bereiter-Hahn J. Strebhardt K. Kuhl D. EMBO J. 1999; 18: 5528-5539Crossref PubMed Scopus (182) Google Scholar, 8Newton S.S. Collier E.F. Hunsberger J. Adams D. Terwilliger R. Selvanayagam E. Duman R.S. J. Neurosci. 2003; 23: 10841-10851Crossref PubMed Google Scholar), leading to degradation of SPAR, a mechanism recently shown to be critical in activity-dependent synaptic scaling (a principal form of homeostatic plasticity) (9Turrigiano G. Curr. Opin. Neurobiol. 2007; 17: 318-324Crossref PubMed Scopus (245) Google Scholar, 43Seeburg D.P. Feliu-Mojer M. Gaiottino J. Pak D.T. Sheng M. Neuron. 2008; 58: 571-583Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). SPAR turnover depends upon the ubiquitin-proteasome pathway (UPP) since ubiquitinated SPAR accumulates in the presence of active Plk2 when proteasomes are inhibited (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). This necessitates the involvement of at least one E3 ubiquitin (Ub) ligase targeting SPAR. More generally, the UPP is known to play an important role in the activity-dependent turnover of several proteins in the PSD (10Ehlers M.D. Nat. Neurosci. 2003; 6: 231-242Crossref PubMed Scopus (843) Google Scholar), a process that likely involves activity-driven redistribution of proteasomes into spines (11Bingol B. Schuman E.M. Nature. 2006; 441: 1144-1148Crossref PubMed Scopus (275) Google Scholar). However, as is the case for many neuronal processes in which the UPP has been implicated, the molecular and regulatory components upstream of the proteasome responsible for targeting the ubiquitination of SPAR and other PSD proteins are unknown. Skp1/Cul1/F-box protein (SCF) complexes are one of the best understood E3 Ub-ligases with regard to mechanism of substrate recognition (12Cardozo T. Pagano M. Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (903) Google Scholar, 13Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1734) Google Scholar). By recruiting substrates to the SCF complex, F-box proteins dramatically increase the specificity and rate of ubiquitin transfer to substrates. The F-box protein β-TRCP has been shown to target critical signaling proteins for degradation, including IκBα, β-catenin, and Cdc25A (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 15Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar, 16Margottin-Goguet F. Hsu J.Y. Loktev A. Hsieh H.M. Reimann J.D. Jackson P.K. Dev. Cell. 2003; 4: 813-826Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 17Winston J.T. Strack P. Beer-Romero P. Chu C.Y. Elledge S.J. Harper J.W. Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (827) Google Scholar, 18Busino L. Donzelli M. Chiesa M. Guardavaccaro D. Ganoth D. Dorrello N.V. Hershko A. Pagano M. Draetta G.F. Nature. 2003; 426: 87-91Crossref PubMed Scopus (372) Google Scholar). Its function in neurons, however, has remained unexplored. Here, we identifyβ-TRCP as the F-box protein that targets Plk2-phosphorylated SPAR for degradation in neurons. Biochemical studies revealed that SPAR interacted with the SCFβ-TRCP complex through a canonical β-TRCP phosphodegron. Induction of Plk2 activity led to SPAR turnover, and this was prevented by dominant negative disruption of the SCFβ-TRCP complex or point mutations in the phosphodegron of SPAR preventing SPAR from interacting with β-TRCP. DNA Plasmids—Expression plasmids expressing cDNA with a CMV promoter for myc-SPAR, HA-Plk2, and HA-Plk2K108M were previously described (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). pCMV-HA-Plk2D201A was created from pCMV-HA-Plk2 using PCR-based mutagenesis. cDNA for full-length SPAR as well as SPAR fragments were amplified from pGW1-myc-SPAR (5Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar) and cloned into pENTR-6, compatible with the GATEway cloning system (Invitrogen). These were used for in vitro LR clonase reactions into pDEST-53 (for GFP-SPAR) or pDEST-N-myc (for myc-SPAR fragments). Point mutations in the β-TRCP phosphodegron were generated by PCR-based mutagenesis using pCMV-myc-SPAR or pDEST-myc-Act2 as templates. pCMV-myc-CKIϵ and pCMV-GSK3β were previously described (19Shirogane T. Jin J. Ang X.L. Harper J.W. J. Biol. Chem. 2005; 280: 26863-26872Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). F-box proteins previously cloned from cDNA pools (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar) were re-cloned into pENTR-6, and in vitro LR clonase reactions were performed with pDEST-27 to generate GST-fused F-box proteins. DNCul1 (residues 1-452) was previously described (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar); DNCul3 and DNCul4 were prepared by cloning sequences encoding residues 1-418 of Cul3 and 1-440 of Cul4A into pcDNA3 (Invitrogen), respectively. β-TRCP1ΔF, Fbw7ΔF, and Skp2ΔF plasmids were previously described (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 20Bashir T. Dorrello N.V. Amador V. Guardavaccaro D. Pagano M. Nature. 2004; 428: 190-193Crossref PubMed Scopus (408) Google Scholar, 21Welcker M. Orian A. Jin J. Grim J.E. Harper J.W. Eisenman R.N. Clurman B.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9085-9090Crossref PubMed Scopus (705) Google Scholar), and β-TRCP2ΔF was a gift from N. Khidekel (MIT). pRetroSuper (pRS)-shβ-TRCP and control shGFP plasmids were previously published (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar). HEK293T Transfections, Binding, Abundance, and Turnover Assays—HEK293T cells were grown in Dulbecco's modified Eagle's medium + 10% serum and seeded (1 × 106 cells/well of a 6-well dish) 16 h before transfection with Lipofectamine 2000 (Invitrogen). Cells were typically harvested ∼24-30 h post-transfection, except for RNAi experiments where cells were harvested 96 h post-transfection after a 48-h selection with puromycin (1 μg/ml) to enrich for a transfected population. Treatment of transfected cells with cycloheximide (25 μg/ml) began 96 h post-transfection. For assays examining myc-SPAR abundance and turnover, cell pellets were lysed in 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, and cleared lysates were resolved on 4-12% gradient Tris-glycine SDS-PAGE gels. Resolved proteins were then transferred onto nitrocellulose (250 mA, 2 h) and immunoblotted with c-Myc 9E10 (sc-40, Santa-Cruz), HA F-7 (sc-7392, Santa Cruz), Cdk2 M2 (sc-163, Santa Cruz), β-TRCP1 (Cell Signaling), Cul1 (71-8700, Invitrogen/Zymed Laboratories Inc.), and Cdc25A Ab-3 (MS-640-P0, NeoMarkers), as indicated. Promega horseradish peroxidase-conjugate anti-mouse IgG (W402B) and anti-rabbit IgG (W401B) were used for secondary detection. For single-cell immunofluorescence RNAi experiments, HEK293T cells were seeded onto 18-mm glass coverslips coated with poly-d-lysine (30 μg/ml) and laminin (2 μg/ml) and transfected at 20-30% confluency using calcium phosphate. Cells were fixed 96 h post-transfection for 10 min at room temperature using 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline, and the GFP-SPAR signal was amplified using anti-GFP (A-11122, Invitrogen) at 1:1000 in GDB buffer (1% gelatin, 5% Triton X-100, 50 mm phosphate buffer, pH 7.4, 2 m NaCl) (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). Binding and ubiquitination detection assays were performed with cleared whole cell lysates from transfected HEK293T cells in 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5% deoxycholate, and 1% Nonidet P-40. In the case of ubiquitination detection assays, cells were treated with 25 μm MG-132 before harvesting, and 10 mm N-ethylmaleimide was added to the lysis buffer. GSH-Sepharose or c-Myc 9E10-agarose beads (10 μl per condition) were pre-washed in lysis buffer and incubated with 400 μg of lysate for 2 h at 4 °C while rocking. Beads were then washed 3-4 times in lysis buffer, and bound protein was eluded in 2× Laemmli protein loading buffer containing SDS. Both GSH-bound samples and crude extract were resolved on 4-12% gradient SDS-PAGE gels. Myc-bound samples were resolved on 6% Tris-Glycine SDS-PAGE gels. Samples were then transferred from the SDS-PAGE onto nitrocellulose and immunoblotted with anti-Myc, anti-HA, anti-FLAG (F3165, Sigma), or anti-GST (26H1, Cell Signaling), as indicated. Neuron Cultures and Immunostaining—Medium-density dissociated hippocampal cultures were prepared and cultured from E19 Long Evans rat hippocampi as previously described (5Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Neurons were transfected at DIV16, "super-infected" at DIV18, and fixed ∼18 h post-infection (DIV19) in 1% paraformaldehyde for 2 min at room temperature followed by -20 °C methanol for 10 min. Immunostaining was performed in GDB buffer (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar) using rabbit SPAR polyclonal antibodies (5Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar) and anti-FLAG M2 (Sigma). Microscopy and Quantification—Fixed neurons and HEK293T cells were imaged with an LSM510 confocal system (Zeiss). A 40× oil immersion lens was used for confocal microscopy, and each image was comprised of 0.5-μm z-stacks projected into a single plane. SPAR immunostaining analysis was performed with MetaMorph Software and carried out blinded with respect to the experimental conditions. Quantification of SPAR puncta involved subjecting images stained for endogenous SPAR to threshold and was carried out from somatic and proximal dendritic regions from transfected, infected, or transfected plus superinfected cells. All SPAR intensity measurements represent integrated SPAR immunostaining intensity per area and are normalized to neighboring uninfected and untransfected cells. Statistical Analysis—Statistical Methods are described in the figures legends. Plk2-dependent SPAR Degradation Requires a Cul1-based E3 Ub-ligase—SPAR turnover in neurons is controlled through the UPP in a manner that requires active Plk2 protein kinase (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). Such regulation is reminiscent of the mechanism employed by most SCF E3 Ub-ligases, where substrate recognition depends upon upstream kinase signaling cascades. A central subunit of the SCF complex is the Cul1 scaffold protein. To explore the idea that SPAR turnover is regulated by an SCF E3 Ub-ligase, we made use of a previously reported dominant-negative version of Cul1 (DNCul1). This C-terminal-truncated protein binds substrates but fails to associate with ubiquitin-charged E2 ubiquitin conjugating enzymes, thereby preventing substrate turnover (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 19Shirogane T. Jin J. Ang X.L. Harper J.W. J. Biol. Chem. 2005; 280: 26863-26872Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 22Piva R. Liu J. Chiarle R. Podda A. Pagano M. Inghirami G. Mol. Cell. Biol. 2002; 22: 8375-8387Crossref PubMed Scopus (47) Google Scholar). Cultured hippocampal neurons (16 days in vitro (DIV16)) were transfected with DNCul1 and then super-infected 2 days later with Sindbis virus driving expression of FLAG-tagged Plk2 for ∼18 h to promote degradation of endogenous SPAR (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). We infected at a titer that resulted in ∼10% infection rate of cells already transfected with DNCul1 (Fig. 1, A-C). The option of co-transfecting plasmids driving Plk2 expression was precluded by the low Plk2 expression achievable using this method. 6D. P. Seeburg, unpublished observation. In the absence of Plk2 infection, transfection of control empty vector (pcDNA3.1) or a control dominant-negative Cullin 3 (DNCul3), which disrupts structurally related but functionally distinct Cul3-based complexes (23Cullinan S.B. Gordan J.D. Jin J. Harper J.W. Diehl J.A. Mol. Cell. Biol. 2004; 24: 8477-8486Crossref PubMed Scopus (797) Google Scholar), had no effect on endogenous SPAR levels relative to nearby untransfected and uninfected cells as assessed by quantitative immunostaining (Fig. 1, A and C, quantified in D). Uninfected cells overexpressing DNCul1 displayed a trend toward increased SPAR levels compared with nearby untransfected cells, but did not reach statistical significance (p = 0.13; Fig. 1B, quantified in D). In Plk2-infected but otherwise untransfected neurons, endogenous SPAR levels were close to undetectable (Fig. 1, A-C, arrowheads; quantified in E), in agreement with our previous findings (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar). In Plk2-infected cells that had been previously transfected with control empty vector or DNCul3, SPAR levels fell to the same extent as in cells only infected with Plk2 (Fig. 1, A and C, compare SPAR staining in cells marked by arrowheads (infected) and arrows (transfected and infected); quantified in E). However, in cells transfected with DNCul1, infection with Plk2 Sindbis virus failed to reduce SPAR levels (Fig. 1B, notice the yellow color in cells marked by arrows, indicating the presence of both SPAR (green) and Plk2 (red); quantified in E). Thus, Plk2-driven SPAR degradation in neurons depended upon a Cul1-based SCF complex but not any of the structurally related Cul3-based Ub-ligases. SPAR Physically Associates with the SCFβ-TRCP Complex—To further explore the idea that SPAR turnover is regulated by an SCF E3 Ub-ligase, we established a system in cultured HEK293T cells that recapitulates Plk2-dependent SPAR degradation. This system facilitated biochemical studies that were otherwise limited by the physical properties of dendritic spines and allowed the use of molecular reagents previously developed for study of the human SCF pathway (24Jin J. Ang X.L. Shirogane T. Wade Harper J. Methods Enzymol. 2005; 399: 287-309Crossref PubMed Scopus (80) Google Scholar). We found that expression of SPAR alone (as an Myc-tagged fusion protein) led to its accumulation in HEK293T cells (Fig. 2A, lane 3). In contrast, co-expression with Plk2 (but not a catalytically inactive mutant Plk2D201A) promoted the degradation of myc-SPAR (Fig. 2, A, lane 2 compared with lanes 4-6, and B, lanes 1 and 2). Co-expression of SPAR with wild type Plk2 correlated with the appearance of a slower mobility form of SPAR (presumably phosphorylated) that is sensitive to Plk2-induced degradation (Fig. 2A, myc-SPAR-P; see also Fig. 2, B and C). Our recapitulation of Plk2-dependent SPAR turnover in HEK293T cells provides a convenient system in which to search for components of the Plk2-dependent degradation pathway. Consistent with the idea that SPAR is a target of one of the SCF complexes, overexpression of dominant-negative Cul1 (DNCul1) in HEK293T stabilized myc-SPAR despite cotransfection of active Plk2 (Fig. 2B, lane 3), in agreement with our findings in neurons (see Fig. 1, B and E). Moreover, these cells accumulated the slower migrating form of myc-SPAR that presumably corresponds to phosphorylated SPAR. In contrast, a dominant-negative form of the related Cullin4 (DNCul4) had no effect on SPAR turnover (lane 4), indicating that the effect of DNCul1 was specific. F-box proteins serve as substrate receptors in Cul1-based E3s. To uncover candidate F-box proteins for SPAR, we performed a cell-based interaction screen in HEK293T cells between SPAR and a panel of co-expressed GST-tagged F-box proteins. Potential complexes between SPAR and GST-tagged F-box proteins were isolated by incubating cell lysates with GSH-Sepharose beads. Previous studies had indicated that the interaction of F-box proteins with substrates can be detected in tissue culture cells that co-express the F-box protein and substrate in the presence of DNCul1, which blocks substrate degradation (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 19Shirogane T. Jin J. Ang X.L. Harper J.W. J. Biol. Chem. 2005; 280: 26863-26872Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Among a panel of F-box proteins individually co-transfected with myc-SPAR and HA-Plk2, we identified an interaction between myc-SPAR and both GST-β-TRCP1 and GST-β-TRCP2 (Fig. 2C, lanes 3 and 4). Our ability to assay for endogenous interactions was precluded by the unavailability of suitable antibodies for immunoprecipitation. Although produced from different genes, β-TRCP1 and β-TRCP2 are ∼85% identical and are believed to have largely redundant functions (25Guardavaccaro D. Kudo Y. Boulaire J. Barchi M. Busino L. Donzelli M. Margottin-Goguet F. Jackson P.K. Yamasaki L. Pagano M. Dev. Cell. 2003; 4: 799-812Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Importantly, the binding of β-TRCP1 and β-TRCP2 to myc-SPAR required co-transfection of active (wild type (WT)) Plk2 and was undetectable in the presence of catalytically defective Plk2K108M (Fig. 2C, lanes 5 and 6). Together with the Plk2-dependent slower gel mobility of SPAR (Fig. 2, A-C), this suggests that SPAR associates with β-TRCP in a phosphorylation-dependent manner, as is the case for all other β-TRCP substrates identified to date (13Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1734) Google Scholar). SPAR did not associate with any of 16 other F-box proteins tested (Fig. 2C, lanes 7-22), including 5 F-box proteins, which like β-TRCP, bind substrates through their WD40 repeats (Fig. 2C, lanes 7-11). This indicates a high degree of specificity in the interaction between SPAR and β-TRCP. Consistent with previous studies that showed interaction between Plk2 and SPAR (6Pak D.T. Sheng M. Science. 2003; 302: 1368-1373Crossref PubMed Scopus (266) Google Scholar), we found that Plk2 associated with SPAR in a coimmunoprecipitation assay upon co-expression of DNCul1 (Fig. 2D), further validating our heterologous cell system. Interestingly, we also discovered that the associations of Plk2 and β-TRCP with SPAR were not mutually exclusive, as we were able to detect a ternary interaction with Plk2 that was not precluded by the β-TRCP-SPAR interaction (Fig. 2E, lanes 6 and 8). GST-β-TRCP1 (and GST-β-TRCP2) associated with SPAR, Plk2, and the N terminus of Cullin 1 (Cul11-452/DNCul1), whereas catalytically inactive Plk2D201A did not support assembly of the full complex (Fig. 2E, lanes 5-8). Moreover, the Plk2-dependent slower mobility form of SPAR was enriched in the complex compared with the faster mobility form of SPAR (Fig. 2E, compare lanes 2 and 4 with 6 and 8). SCFβ-TRCP Regulates SPAR Abundance and Turnover and Promotes Its Ubiquitination—To validate a role for the SCFβ-TRCP complex in Plk2-dependent SPAR turnover, we next examined SPAR abundance in HEK293T cells after expression of dominant-negative β-TRCP (β-TRCPΔF) (Fig. 2B). The ΔF-box construct is unable to assemble with Cul1 due to absence of the F-box motif, but it maintains its ability to interact with substrates of both β-TRCP1 and -2 and can thereby sequester substrates and block their turnover (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 21Welcker M. Orian A. Jin J. Grim J.E. Harper J.W. Eisenman R.N. Clurman B.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9085-9090Crossref PubMed Scopus (705) Google Scholar, 24Jin J. Ang X.L. Shirogane T. Wade Harper J. Methods Enzymol. 2005; 399: 287-309Crossref PubMed Scopus (80) Google Scholar). Expression of β-TRCPΔF resulted in increased levels of SPAR, especially of the slower migrating form of SPAR that is dependent upon Plk2 activity (Fig. 2B, lane 5). In contrast, ΔF-box dominant-negative versions of other F-box proteins Fbw7α/β/γ (containing WD40 repeats) and Skp2 (containing leucine-rich repeats) failed to promote an increase in the steady state abundance of SPAR (Fig. 2B, lanes 6-9). To directly examine whether β-TRCP proteins are required for Plk2-dependent SPAR turnover, we took advantage of a shRNA vector (shβ-TRCP) that is capable of suppressing protein expression of both human β-TRCP1 and β-TRCP2. This hairpin sequence and this particular shRNA vector have been validated for numerous β-TRCP substrates (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 19Shirogane T. Jin J. Ang X.L. Harper J.W. J. Biol. Chem. 2005; 280: 26863-26872Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 26Fong A. Sun S.C. J. Biol. Chem. 2002; 277: 22111-22114Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Cells were transfected with expression constructs for GFP-SPAR and red fluorescent protein (RFP) to mark transfected cells and simultaneously transfected with shβ-TRCP or control vector (pRSP) in the presence of active Plk2 or a catalytically inactive version, Plk2D201A (Fig. 3A). We subsequently visualized cells for the presence of GFP-SPAR in RFP-positive cells (Fig. 3A). RFP-positive cells expressing Plk2, but not those expressing catalytically inactive Plk2D201A, displayed very low levels of GFP-SPAR in the presence of the control shRNA vector pRSP (Fig. 3A, quantified in B). In contrast, RFP-positive cells transfected with shβ-TRCP contained high levels of GFP-SPAR despite the co-transfection of active Plk2 (Fig. 3A, quantified in B). Thus, depletion of β-TRCP by RNAi substantially protected GFP-SPAR from Plk2-dependent degradation. Immunoblotting of transfected cell lysates confirmed that Plk2-induced degradation of myc-SPAR requires β-TRCP (Fig. 3C). As expected, Plk2 promoted loss of myc-SPAR in cells expressing a control shRNA that targets GFP (shGFP) (lanes 1 and 2). In contrast, myc-SPAR was not efficiently degraded in cells depleted of β-TRCP despite the presence of active Plk2 (lane 4). Furthermore, depletion of β-TRCP led to accumulation of Cdc25A, a known target of SCFβ-TRCP used here as a positive control (14Jin J. Shirogane T. Xu L. Nalepa G. Qin J. Elledge S.J. Harper J.W. Genes Dev. 2003; 17: 3062-3074Crossref PubMed Scopus (291) Google Scholar, 18Busino L. Donzelli M. Chiesa M. Guardavaccaro D. Ganoth D. Dorrello N.V. Hershko A. Pagano M. Draetta G.F. Nature. 2003; 426: 87-91Crossref PubMed Scopus (372) Google Scholar). To directly examine whether β-TRCP is required for SPAR turnover, we performed a cycloheximide-chase experiment in cells expressing myc-SPAR and Plk2 in the presence of shGFP or shβ-TRCP (Fig. 3D). Myc-SPAR levels persisted after 45-60 min of cycloheximide treatment under conditions of β-TRCP depletion (shβ-TRCP) but disappeared after the same time period in control cells (shGFP) (Fig. 3D, lanes 7-8 compared with 3 and 4). Taken together, these data indicate that SCFβ-TRCP is critical for Plk2-dependent degradation of SPAR in heterologous cells. To explore whether the SCFβ-TRCP complex promoted ubiquitination of SPAR, we immunoprecipitated myc-SPAR from MG-132-treated HEK293T cells and immunoblotted for modified myc-SPAR (Fig. 3E). Expression of active Plk2 resulted in detection of ubiquitinated SPAR, and the ubiquitination reaction was further driven in cells by ectopic expression of GST-β-TRCP (lanes 2 and 3). Together, this suggests that the Plk2-dependent turnover of myc-SPAR occurs through ubiquitination of SPAR. Plk2-dependent Recognition of SPAR by β-TRCP Involves a Canonical Phosphodegron—SPAR is a large protein of 1804 amino acids containing two actin binding domains (Act1 and Act2), a RapGAP domain, a PDZ domain, and a guanylate kinase binding domain (GKBD) (Fig. 4A). Within the Act2 domain, we identified a candidate β-TRCP recognition motif (DSGIDT, residues 1304-1309) based upon the consensus β-TRCP recognition motif found in many of its targets (DpSGΦX(pS/T); Φ= hydrophobic residue, X = any residue, pS or pS/T = phosphoserine or threonine) (Fig. 4A). Initially,
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