Supplemental Information. Phosphorylation by Casein Kinase I Promotes. the Turnover of the Mdm2 Oncoprotein. via the SCF β-trcp Ubiquitin Ligase

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1 Cancer Cell, Volume 18 Supplemental Information Phosphorylation by Casein Kinase I Promotes the Turnover of the Mdm2 Oncoprotein via the SCF β-trcp Ubiquitin Ligase Hiroyuki Inuzuka, Alan Tseng, Daming Gao, Bo Zhai, Qing Zhang, Shavali Shaik, Lixin Wan, Xiaolu L. Ang, Caroline Mock, Haoqiang Yin, Jayne M. Stommel, Steven Gygi, Galit Lahav, John Asara, Zhi-Xiong Jim Xiao, William G. Kaelin, Jr., J. Wade Harper, and Wenyi Wei SUPPLEMENTAL SECTION INVENTORY SUPPLEMENTAL FIGURES Figure S1 relates to manuscript Figure 1. Figure S2 relates to manuscript Figure 2. Figure S3 relates to manuscript Figure 3. Figure S4 relates to manuscript Figure 4. Figure S5 relates to manuscript Figure 5. Figure S6 relates to manuscript Figure 6. Figure S7 relates to manuscript Figure 7. SUPPLEMENTAL EXPERIMENTAL PROCEDURES SUPPLEMENTAL REFERENCES

2 Figure S1, related to Figure 1. Mdm2 stability is controlled by β-trcp A. Immunoblot analysis of HeLa cells transfected with the indicated sirna oligos. B. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Myc-tagged Cullin 1 and the indicated HA-Mdm2 constructs. Twenty hours post-transfection, cells were treated with 10 µm MG132 overnight before harvesting. C. Immunoblot analysis of wild-type and β-trcp1 -/- mouse embryonic fibroblasts (MEFs). D. Immunoblot analysis of 293T cells transfected with the indicated shrna constructs together with HA-Mdm2 and jellyfish GFP (egfp, as transfection controls). Where indicated, Flag-β-TRCP1**, which is engineered to resist the shβ-trcp1+2 effect, was included in the transfection. E. Immunoblot analysis of HeLa cells transfected with the indicated sirna oligos, after synchronization with nocodazole and release.

3 Figure S2, related to Figure 2. Mdm2 C464A binds to β-trcp1 as efficiently as wild-type Mdm2 Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Myc-tagged β-trcp1 and the indicated HA-Mdm2 constructs. Twenty hours posttransfection, cells were treated with 10 µm MG132 overnight before harvesting.

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6 Figure S3, related to Figure 3. Casein Kinase I is involved in regulating Mdm2 stability A. Illustration of the identified major PEST sequences within the Mdm2 primary protein sequence using the PESTfinder program. B. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Mdm2 and various Myc-tagged CKI constructs. Twenty hours posttransfection, cells were treated with 10 µm MG132 overnight before harvesting. C. Autoradiograms showing various recovered 35 S-labeled CKI isoforms bound to GST-Mdm2 fusion proteins (GST protein as a negative control). IN, input (10% or 2% as indicated). D. Immunoblot analysis of whole cell lysates (WCL) derived from HeLa cells transfected with HA- Mdm2 and various amounts of Myc-tagged CKIδ constructs. E. Immunoblot analysis of whole cell lysates (WCL) derived from HeLa cells transfected with HA- Mdm2 and the indicated Myc-tagged CKIδ constructs. The K38R-CKIδ construct is a kinasedead mutant of CKIδ, which is used as a negative control in this assay. F. Immunoblot analysis of whole cell lysates (WCL) derived from HeLa cells transfected with the indicated Myc-tagged CKIδ constructs to detect the changes in endogenous Mdm2 expression. The K38R-CKIδ construct is a kinase-dead mutant of CKIδ, which is used as a negative control in this assay. G. HeLa cells were transfected with HA-Mdm2 and the indicated Myc-tagged CKIδ constructs. Twenty hours post-transfection, cells were split into 60 mm dishes, and after another 20 hours, cells were treated with 20 μg/ml cycloheximide. At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies. H. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Mdm2 and Myc-tagged β-trcp1 constructs. Where indicated, cells were treated with two different CKI inhibitors (25 μm IC261 and 15 μm D4476) for 6 hours

7 before harvesting. Twenty hours post-transfection, cells were treated with 10 µm MG132 overnight before harvesting. I. Immunoblot analysis of HeLa cells transfected with HA-Mdm2 and the indicated shrna constructs. J. HeLa cells were infected with the indicated lentiviral shrna construct and selected with 1 µg /ml puromycin to eliminate non-infected cells. The resulting HeLa cell lines were arrested in the M phase by incubation with nocodazole for 18 hours and then released into the G1 phase. At the indicated time points, cell lysates were collected for immunoblot analysis. K. HeLa cells were transfected with the shgfp or shckiδ plasmid. Twenty hours post-transfection, cells were split into 60 mm dishes. After another 20 hours, cells were treated with 20 μg/ml cycloheximide. At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies. L. Quantification of the band intensities in K. Mdm2 band intensity was normalized to tubulin, then normalized to the t=0 controls. M. Immunofluorescence and DAPI staining of U2OS cells treated with or without 10 µm doxorubicin for 1 hour. Scale bars: 10 μm. N. Quantitation of the percentage of cells with nuclear CKIδ staining in M. The error bars represent +/- SD.

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13 Figure S4, related to Figure 4. Casein Kinase I phosphorylates Mdm2 at multiple sites to trigger Mdm2 interaction with β-trcp1 A. Schematic illustration of the Mdm2 protein functional domains and the identified PEST sequences (p1, p2 and p3 region). B. Schematic representation of in vitro phosphorylation of GST-Mdm2 by CKIδ detected by mass spectrometry analysis. Confidentially assigned phosphorylation sites are shown in green while phosphorylation sites shown in red are highly possible phosphorylation sites, but not confidently assigned. C. Schematic representation of in vivo Mdm2 phosphorylation sites in 293T cells treated with the proteasomal inhibitor MG132 (with DMSO as a negative control) detected by mass spectrometry analysis. Confidentially assigned phosphorylation sites are shown in green while phosphorylation sites shown in red are highly possible phosphorylation sites, but not confidently assigned. D. Illustration of the reported CKI phosphorylation sites in Mdm2. E. Illustration of the verified and predicted Mdm2 phosphorylation sites that mimic the β-trcp degron sequence. These potentially critical Ser/Thr phosphorylation sites for individual suboptimal degrons including the putative CKI sites were mutated in a step-wise fashion, and their abilities to be phosphorylated by CKIδ, to interact with and to be degraded by β-trcp1 were analyzed in Figure 4-5. Verified phosphorylation sites are shown in green while predicted phosphorylation sites are shown in blue. The surrounding amino acids, which are possible components of the putative degron sequence are also highlighted in orange. F. Sequence alignment of the putative β-trcp-recognizable non-canonical degron sequence in the p1 region of Mdm2 across different species. G. HeLa cells were transiently transfected with the HA-tagged wild-type or a mutant (S118A/S121A) Mdm2 construct. Thirty hours post-transfection, Mdm2 was recovered by HAimmunoprecipitation, and probed with an antibody that could detect the phosphorylation status of

14 Ser118 or Ser118/Ser121. Where indicated, cells were treated with 15 µm MG132 overnight before harvesting. H. HeLa cells were treated with or without the proteasome inhibitor MG132 to block Mdm2 destruction. Endogenous Mdm2 was immunoprecipitated with anti-mdm2 antibody (with mouse IgG as a negative control). Where indicated, whole cell lysates were treated with λ-phosphatase prior to immunoprecipitation. The recovered Mdm2 immunoprecipitate was separated by SDS- PAGE and immunoblotted with an antibody that could detect the phosphorylation status of Ser118. I. HeLa cells were transiently transfected with the HA-tagged Mdm2 construct. Thirty hours posttransfection, the Mdm2 protein was recovered by HA-immunoprecipitation, and probed with an antibody that could detect the phosphorylation status of Ser118/Ser121. Where indicated, cells were treated with 50 µm etoposide for the indicated times before harvesting. Cells were treated with 10 µm MG132 for 1 hour prior to DNA damage treatment to block Mdm2 destruction. J. HeLa cells were treated with or without the proteasome inhibitor MG132 to block Mdm2 destruction. Endogenous Mdm2 was immunoprecipitated with anti-mdm2 antibody (with mouse IgG as a negative control). Where indicated, cells were treated with the CKI inhibitor D4476 (15 μm) for 3 hours prior to harvesting for immunoprecipitation. The recovered Mdm2 immunoprecipitate was separated by SDS-PAGE and immunoblotted with an antibody that could detect the phosphorylation status of Ser118. K. 293T cells were transiently transfected with HA-Mdm2 and the indicated shrna constructs. Thirty hours post-transfection, Mdm2 was recovered by HA-immunoprecipitation, and probed with an antibody that could detect the phosphorylation status of Ser118/Ser121. L. Sequences of the biotinylated Mdm2 peptides used in the β-trcp binding assays. M-N. Autoradiograms showing recovery of 35 S-labeled F-box proteins bound to the indicated biotinylated peptides. IN, input (10%, 5% or 2% as indicated).

15 O-P. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Flag-β-TRCP1 and the indicated HA-Mdm2 constructs. Twenty hours post-transfection, cells were treated with 10 µm MG132 overnight before harvesting. Q. Schematic illustration of the various Mdm2 deletion mutants generated for this study. R. Illustration of the various Mdm2 mutants with gradual inactivation of the potentially critical Ser/Thr phosphorylation sites for suboptimal degrons. The abilities of these individual mutants to be phosphorylated by CKIδ and degraded by β-trcp were further analyzed in Figure 4-5. S. CKIδ phosphorylates Mdm2 in vitro at multiple sites. Purified CKIδ protein (from New England Biolabs) was incubated with 5 μg of the indicated GST-Mdm2 proteins in the presence of γ- 32 P- ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography. T. Point-mutations in Mdm2 do not affect its interaction with CKIδ in vitro. Whole cell lysates (WCL) derived from 293T cells transfected with Myc-CKIδ were incubated with 3 μg of the indicated GST-Mdm2 proteins. After intensive washing, the recovered GST precipitations were resolved by SDS-PAGE and immunoblot was performed with anti-myc antibody. The PVDF membrane was stained with Ponceau S to indicate equal loading of the indicated GST-proteins on each lane. U. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated HA-Mdm2 and Myc-tagged CKI constructs. Twenty hours post-transfection, cells were treated with 10 µm MG132 overnight before harvesting. V. Phosphorylation of Mdm2 at multiple sites by CKIδ triggers its interaction with β-trcp1 in vitro. Autoradiograms showing recovery of 35 S-labeled β-trcp1 protein bound to the indicated GST- Mdm2 fusion proteins (GST protein as a negative control) incubated with CKIδ prior to the pulldown assays. IN, input (10% or 2% as indicated).

16 Figure S5, related to Figure 5. GST-p1 and GST-p2 were susceptible to β-trcp1-mediated proteolysis Immunobot analysis of HeLa cells transfected with the indicated GST-Mdm2 and shrna plasmids. A plasmid encoding GFP was used as a negative control for transfection efficiency.

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18 Figure S6, related to Figure 6. Casein Kinase Iδ phosphorylates Mdm2 at multiple sites to trigger Mdm2 ubiquitination and destruction by β-trcp1 A. HeLa cells were transfected with the indicated HA-Mdm2 constructs. Twenty hours posttransfection, cells were split into 60 mm dishes, and after another 20 hours, treated with 20 μg/ml cycloheximide. At the indicated time points, whole-cell lysates were prepared and immunoblots were probed with the indicated antibodies. B. HeLa cells were transfected with the indicated HA-Mdm2 constructs together with the Flag-β- TRCP1 and Myc-CKIδ plasmids. Twenty hours post-transfection, cells were split into 60 mm dishes, and after another 20 hours, treated with 20 μg/ml cycloheximide. At the indicated time points, whole-cell lysates were prepared and immunoblots were probed with the indicated antibodies. C. Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated HA-Mdm2 and GST-p53 constructs. Twenty hours posttransfection, cells were treated with 10 µm MG132 overnight before harvesting. D. Mutation of the identified major Ser/Thr phosphorylation sites does not affect the ability of Mdm2 to degrade p53 while deletion of the p1, p2 and p3 PEST sequences moderately reduces the E3 ligase activity of Mdm2 towards p53 destruction. HeLa cells were transfected with the indicated HA-Mdm2 constructs. Forty hours post-transfection, whole-cell lysates were prepared and immunoblots were probed with the indicated antibodies. E. Immunoblot analysis of whole cell extracts and anti-ha immunoprecipitates of HeLa cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight, and stimulated with 10 μm etoposide for 2 hours before harvesting.

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21 Figure S7, related to Figure 7. β-trcp1 depletion-induced resistance to apoptosis is partially through the p53 pathway and inhibition of Casein Kinase Iδ activity attenuated the p53 pulse in response to continuous DNA damage treatment A. Immunoblot analysis of HeLa cells transfected with HA-Mdm2 and the indicated shrna constructs. Twenty hours post-transfection, cells were treated with etoposide (or with DMSO as a negative control) for 1.5 hours. B. Immunoblot analysis of the indicated HeLa stable cell lines transfected with the indicated shrna constructs. Twenty hours post-transfection, cells were treated with etoposide (or with DMSO as a negative control) for 1.5 hours. The indicated HeLa stable cell lines were generated by infection with the shgfp or shckiδ lentiviral constructs and subsequent selection with 1 μg/ml puromycin to eliminate non-infected cells. C-D. U2OS cells were transfected with the indicated sirna oligos. 20 hours post-transfection, cells were treated with hydroxyurea for 16 hours, and then treated with 1.7 µm doxorubicin for 2 hours before release to normal medium. Twelve hours later, cells were recovered for FACS analysis for detection of the apoptotic (sub-g1) cell population (D) or by Annexin V/7-AAD double straining (C). E. Immunoblot analysis of U2OS cells transfected with the indicated sirna oligos to illustrate the knock down efficiency of the anti-p53 sirna oligo used in the assay. F. HCT116-WT or HCT116-p53 -/- cells were infected with the indicated lentiviral shrna constructs. Forty hours post-infection, cells were selected with 1 µg/ml puromycin to create stable cell lines where the endogenous β-trcp1 is depleted (with shgfp as a negative control). The resulting cell lines were treated with hydroxyurea for 16 hours, and then treated with 1.7 µm doxorubicin for 2 hours before release to normal medium. Twelve hours later, cells were recovered for FACS analysis for detection of the apoptotic (sub-g1) cell population.

22 G. Immunoblot analysis of the various generated HCT116 cell lines to illustrate that Mdm2 upregulation by β-trcp1-depletion is independent of the p53 status. H. Real-time RT-PCR analysis to examine the relative β-trcp1 mrna expression levels in U2OS cells transfected with the indicated sirna oligonucleotides. Three independent sets of experiments were performed to generate the error bar. The error bars represent +/- SD. I. Immunoblot analysis of U2OS cells transfected with the indicated sirna oligos. J. Depletion of endogenous β-trcp1 by shtrcp1 treatment (shgfp as a negative control) in HCT116 cells resulted in elevated BrdU incorporation. The indicated HCT116 cells were arrested in M phase with nocodazole incubation and then released to the cell cycle. At the indicated time points post-nocodazole release, cells were pulsed with BrdU for 20 minutes, fixed with ice-cold Methanol and the percentage of BrdU incorporated cells were analyzed with immunohistochemistry. The error bars represent +/- SD. K. Quantitation of bioluminescent imaging studies of xenografted tumors formed by HCT116 cells as in G that were then superinfected with a retrovirus encoding firefly luciferase. A total of 1.5x10 6 cells were injected subcutaneously into the nude mice (shgfp on the left flank and shtrcp1 on the right flank of the individual nude mice). Bioluminescent images were obtained 1 week later (day 0) and serially after the injection. Error bars represent +/- SEM. See Experimental Procedures for normalization. L. Immunoblot analysis of MCF7 cells treated with the CKI inhibitor D4476 (or with DMSO as a negative control) for 7 hours before cells were treated with NCS. Whole cell lysates were collected at the indicated time points. M. MCF7 cells were infected with the indicated lentiviral shrna constructs. Forty hours postinfection, cells were selected with 1 µg/ml puromycin to create stable cell lines where the endogenous CKIδ is depleted (with shgfp as a negative control). The resulting cell lines were treated with NCS, and whole cell lysates were collected at the indicated time points.

23 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Plasmids: HA-Mdm2 construct was obtained from Dr. Jiandong Chen. Mdm2, β-trcp1 and CKIδ mutants were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer s instructions. Various Mdm2 cdna were subcloned using the Pfu polymerase (Stratagene) into the pgex vector to create GST-Mdm2 in frame fusion proteins. Indicated fragments of Mdm2 cdna (Fig. 5D) were subcloned using the Pfu polymerase (Stratagene) into pcmv-gst vector to create constructs that could ectopically overexpress chimera GST-Mdm2 fusion protein in mammalian cells. Flag-β-TRCP1, shrna-β-trcp1+2, shtrcp1, shtrcp2, shrna-gfp and various CKI constructs were described previously (Jin et al., 2003; Shirogane et al., 2005). The Fbw4, Fbw5, Skp2 constructs were described previously (Wei et al., 2005). Myc-β-TRCP1, Myc-β-TRCP2 and Myc-β-TRCP1-R474A constructs were described previously (Jin et al., 2003; Shirogane et al., 2005; Wu et al., 2003). GST-Cullin-1, Myc-Cullin 1, Myc-Cullin 2, Myc-Cullin 3, Myc-Cullin 4A and Myc-Cullin 5 constructs were kind gifts from Dr. James DeCaprio. shrna constructs against various CKI isoforms were obtained from Dr. Jianping Jin. Lentiviral shrna constructs against GFP and CKIδ were obtained from Dr. William Hahn. Antibodies and Reagents: Anti-p21 (sc-397), anti-mdm2 (sc-965), anti c-myc (sc-40), anti-p53 (sc-126), anti-p27 (SC-528), polyclonal anti-ha (SC-805), polyclonal anti-skp2 (SC-7164), anti-cyclin A (SC-751), anti-cyclin B (SC-245), anti- Cdc20 (SC-8358), anti-casein Kinase Iα (SC-6477), anti-casein Kinase Iδ (R-19) (SC-6474), anti-casein Kinase Iδ (H-60) (SC-20709), anti-cullin 1 (SC-17775), anti-plk1 (SC-17783), anti-wee1 (SC-325), anticyclin E (SC-247) antibodies and anti-mdm2 agarose beads (sc-965ac) were purchased from Santa Cruz. Anti-Casein Kinase Iε (610445) was purchased from BD Transduction Laboratory. Anti-tubulin (T-5168), polyclonal anti-flag (F2425), monoclonal anti-flag (F-3165), anti-β-catenin (C7207), anti-vinculin

24 (V4505), peroxidase-conjugated anti-mouse secondary antibody (A4416) and peroxidase-conjugated antirabbit secondary antibody (A4914), anti-flag agarose beads (A2220) and anti-ha agrarose beads (A2095) were purchased from Sigma. Monoclonal anti-ha antibody (MMS-101P) was purchased from Convace. Anti-GFP (632380), monoclonal anti-skp2 ( ) and polyclonal anti-cdh1 ( ) antibodies were purchased from Invitrogen. Monoclonal anti-cdh1 (CC43) was purchased from Oncogene. Anti-Bax (2772), Anti-phospho-Ser317 Chk1 (2344) antibodies were purchased from Cell Signaling. Anti-Mdm2 (Ab1: OP46) and anti-mdm2 (Ab5: OP145) antibodies were purchased from Calbiochem. Anti-Claspin antibody (A267A) was purchased from Bethyl. Polyclonal anit-pser118-mdm2 and anti-pser118pser121-mdm2 antibodies were produced by Proteintech Group, Inc. using synthetic peptides (QESSDSpGTSVSENRC) and (QESSDSpGTSpVSENRC). Oligofectamine, Lipofectamine and Plus reagents were purchased from Invitrogen. Immunoblots and Immunoprecipitation: Cells were lysed in EBC (50 mm Tris ph 8.0, 120 mm NaCl, 0.5% NP-40) buffer supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of the lysates were measured using the Bio-Rad protein assay reagent on a Beckman Coulter DU-800 spectrophotometer. The lysates were then resolved by SDS- PAGE and immunoblotted with the indicated antibodies. For immunoprecipitation, 800 μg lysates were incubated with the appropriate antibody (1-2 μg) for 3-4 hours at 4 C followed by one hour-incubation with Protein A sepharose beads (GE Healthcare). Immuno-complexes were washed five times with NETN buffer (20 mm Tris, ph 8.0, 100 mm NaCl, 1 mm EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. Quantification of the immunoblot band intensity was performed with ImageJ software.

25 Protein Degradation Analysis: Cells were plated into 6 cm tissue-culture dishes 20 hours before transfection. When cells reached an appropriate confluence, they were transfected with 0.4 μg of a plasmid encoding a HA-tagged version of Mdm2 along with 1.0 μg Flag-β-TRCP1, and 0.1 μg of a plasmid encoding GFP as a negative control, in the presence or absence of 0.4 μg of Myc-CKI. For half-life studies, cycloheximide (20 μg/ml, Sigma) was added to the media 40 hours post-transfection. At various time points thereafter, cells were lysed and protein concentration was measured. 30 μg of the indicated whole cell lysates were separated by SDS-PAGE and protein abundances were measured by immunoblot analysis. Detection of Mdm2 phosphorylation sites in vivo: To map Mdm2 phosphorylation status in vivo, 293T cells were transfected with HA-Mdm2 using the calcium phosphate method. Thirty hours post-transfection, 293T cells were treated with 10 μm MG132 for 16 hours to block the 26S proteasome pathway prior to collecting the whole cell lysate for HA-immunoprecipitation. After extensive washing with NETN buffer, the HA-immunoprecipitates were separated by SDS-PAGE and visualized by the Gel-Code Blue reagent. The band containing Mdm2 was excised and treated with DTT to reduce disulfide bonds and iodoacetamide to derivatize cysteine residues. In-gel digestion of the protein was performed with trypsin and chymotrypsin. The resulting peptides were extracted from the gel and analyzed by nanoscale-microcapillary reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) essentially as described previously (Villen and Gygi, 2008). All peptide matches were filtered based on mass deviation, tryptic state, XCorr and dcn and confirmed by manual validation. The reliability of sitelocalization of phosphorylation events was evaluated using the Ascore algorithm (Beausoleil et al., 2006). Detection of Mdm2 phosphorylation sites in vitro: GST-Mdm2 protein purified from BL21 E. Coli was incubated with recombinant CKIδ (from New England Biolabs) to perform in vitro kinase reaction as described above. The reaction was stopped by the addition of

26 SDS-containing lysis buffer, and separated using SDS-PAGE. The gel was stained with the Gel-Code Blue reagent, destained and the Mdm2 band was excised. Samples were subjected to reduction with 10mM dithiothreitol (DTT) for 30 minutes, alkylation with 55mM iodoacetamide with 45 minutes, and in-gel digestion with a combination of chymotrypsin and trypsin enzymes. The digested samples were subjected to reversed phase microcapillary/tandem mass spectrometry (LC/MS/MS) as described previously (Dephoure et al., 2008; Villen and Gygi, 2008), or performed using an EASY-nLC splitless nanoflow HPLC (Proxeon Biosciences) with a self-packed 75 μm id x 15 cm C 18 Picofrit column (New Objective) coupled to a LTQ- Orbitrap XL mass spectrometer (Thermo Scientific) in the data-dependent acquisition and positive ion mode at 300 nl/min with one full MS-FT scan followed by six MS/MS spectra. The analysis of the collected MS/MS spectra data was described previously (Beausoleil et al., 2006; Dephoure et al., 2008; Dibble et al., 2009; Gwinn et al.). Half of the digested peptide pool was reserved for enrichment with the Phos-trap Phosphopeptide Enrichment Kit containing titanium dioxide (TiO 2 ) coated magnetic beads (PerkinElmer) according to the vendor s protocol. Briefly, peptide mixtures containing phosphopeptides were acidified with Binding buffer and incubated with 20µL of 20X TiO 2 magnetic beads diluted in 180µL of HPLC grade water for one hour at room temperature (RT) with continuous shaking in a RT incubator followed by washing three times with Binding buffer and one time with Washing buffer. Phosphopeptides were then incubated with 35µL of basic Elution buffer with continuous shaking. Elution buffer was then transferred to a 12x32mm autosampler vial with 50uL of HPLC A buffer and the final solution was concentrated to 5µL using a SpeedVac prior to injection via LC/MS/MS. Real-time RT-PCR Analysis: RNA was extracted using Qiagen RNeasy mini kit, and the reverse transcription reaction was performed using the ABI Taqman Reverse Transcriptional Reagents (N ). After mixing the resulting template with Mdm2 (Hs _m1), β-trcp1 (Hs _m1) or GAPDH (Hs _m1) primers and ABI Taqman Fast Universal PCR Master Mix ( ), the real-time RT-PCR reaction was performed with the ABI-7500 Fast Real-time PCR system.

27 SUPPLEMENTAL REFERENCES Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J., and Gygi, S. P. (2006). A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24, Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J., and Gygi, S. P. (2008). A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A 105, Dibble, C. C., Asara, J. M., and Manning, B. D. (2009). Characterization of Rictor phosphorylation sites reveals direct regulation of mtor complex 2 by S6K1. Mol Cell Biol 29, Gwinn, D. M., Asara, J. M., and Shaw, R. J. Raptor is phosphorylated by cdc2 during mitosis. PLoS One 5, e9197. Shirogane, T., Jin, J., Ang, X. L., and Harper, J. W. (2005). SCFbeta-TRCP controls clock-dependent transcription via casein kinase 1-dependent degradation of the mammalian period-1 (Per1) protein. J Biol Chem 280, Villen, J., and Gygi, S. P. (2008). The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3,