Supplemental Figure Legends s1

Supplemental Figure Legends

SF1: Fbw7 dimers and monomers interact indistinguishably with cyclin ET62A. A.

35S pulse-chase analyses of cyclin ET62A half-lives when co-transfected with Fbw7 dimers (wt-Fbw7) or monomers (Fbw7 ∆ D ), or with vector control in 293A cells.

Quantified signals were plotted on the right. B. In vitro ubiquitylation of cyclin E or cyclin ET62A with recombinant Fbw7 dimers (D) and monomers (M).

SF2: Alternative Fbw7 dimer-deficient mutants confirm the observations made with Fbw7∆ D. A. Illustration of truncation mutants. M240 lacks the initial essential amino acids of the dimerization domain, but contains all residues missing in

Fbw7∆ D. B. M240 is a monomer. Cells were co-transfected with differentially tagged Fbw7 as indicated and lysates immunoprecipitated and western blotted.

PCNA is a loading control. C. Weak degron positions reveal dimer-dependence with M240 monomer mutant. Cells were transfected and blotted as indicated.

PCNA is a loading control. Assay as in Fig. 2A. D and E. Lack of dimerization and cyclin E/T380S turnover by a double-point mutant Fbw7 (Fbw7LI). Cells were assayed as above.

SF3. Analyses of the cyclin E-Fbw7 interaction. A. Degron analysis of the P-2 position substituting the cyclin E T380 degron with residues naturally found in other substrates in this position (glycine: Jun and PGC1, threonine: SREBP1).

Cells were transfected and blotted as indicated. PCNA is a loading control. B.

Co-immunoprecipitation of central T/S exchanges in both cyclin E degrons with Fbw7 dimers (Fbw7∆ F) and monomers (Fbw7 ∆ FD). 293A cells were co- transfected as indicated, immunoprecipitated with FLAG antibody to Fbw7 and western blotted. C and D. A P+2 proline introduction increases the cyclin ET62

CPD competence. Co-immunoprecipitation analysis (C) of mixed lysates expressing the indicated input (D). E and F. Engineered Fbw7 propeller mutants interact with cyclin E in a dimer-dependent fashion. Assay as in (C and D).

SF4. Generation of dimer-deficient cell lines of endogenous Fbw7. A. Depiction of the exon/intron border of the targeted region in the Fbw7 gene. Yellow: deletion used in FLAG-Fbw7∆ D, red: deletion chosen for gene targeting

(EWLKMF). B. Confirmation the EWLKMF prevents Fbw7 dimerization. 293A cells were co-transfected with differentially tagged Fbw7 and lysates immunoprecipitated and blotted as indicated. Asterisk denotes heavy chain. C.

Illustration of gene targeting strategy used for both AAV vectors (see methods).

D. Depiction of Southern screening strategy to identify correct homozygous gene targeting and excision of Neo cassette, as well as a representative Southern blot.

SF5. Fbw7 monomers and dimers can be distinguished in non-denaturing gels.

Two amounts of recombinant Fbw7 monomers (M) and dimers (D) purified from

SF9 cells were separated on denaturing and non-denaturing gels and stained with Coomassie blue. SF6. Dimer-dependent SREBP-Fbw7 interaction. A. To prevent SREBP turnover, cells were transfected separately with the indicated Fbw7 constructs or with

SREBP1 or 2. Lysates were mixed, immunoprecipitated and blotted as indicated.

Asterisk marks heavy chain. B. SREBP1 binding to Fbw7 requires SREBP1 dimerization in vitro. GST pull-down of in vitro phosphorylated SREBP1 dimers

(WT) and SREBP1 monomers (∆ D) with Fbw7 dimers. C. lysate input for (B).

SF7. Pin1 overexpression does not disrupt Fbw7 dimers. Cells were co- transfected as indicated and blotted on denaturing (upper panels) and non- denaturing gels (lower panel). Pin1 overexpression does not convert Fbw7 dimers to monomers.

SF8. Dimerization regulates Fbw7 auto-ubiquitylation. A. Ectopic Fbw7 half-life is proteasome-dependent. 35S pulse-chase analysis of transfected Fbw7 in the absence or presence of the proteasome inhibitor velcade. Chase times in hrs. B.

Ectopic Fbw7 instability is F-box-dependent. Transfected 293A cells were analyzed by pulse chase, comparing wild-type Fbw7 with an F-box deletion mutant. C. Trans-ubiquitylation of Fbw7 in vitro. 293A cells were co-transfected with MYC-Fbw7F and either FLAG-Fbw7 or FLAG-Fbw7F. Lysates were subjected to sequential immunoprecipitation to isolate MYC- and FLAG-tagged hetero-dimers, followed by an in vitro ubiquitylation reaction and immunoblotted.

The MYC blot was stripped and re-probed with Fbw7-specific antibody to demonstrate the 1:1 ratio of MYC- vs. FLAG-Fbw7. Note that MYC-∆ F and FLAG-Fbw7 co-migrate due to the F-box deletion. D. In vitro auto-ubiquitylation of recombinant Fbw7 monomers (M) and dimers (D) (Rx- reaction mix only). E.

Fbw7F dominantly stabilizes wild-type Fbw7 in a dimer-dependent fashion.

Cells were co-transfected and immunoblotted as indicated. F. Dominant Fbw7 stabilization by co-transfection requires dimerization. MYC-Fbw7 was co- transfected with FLAG-truncation mutants progressively deleting the dimerization domain (+ indicates dimerization) (Welcker and Clurman 2007).

SF9. Fbw7 dimerization facilitates substrate target lysine utilization. In vitro ubiquitylation of cyclin E with recombinant Fbw7 dimers and monomers comparing wt and K48R ubiquitin.

Supplementary methods

Cell culture, transfections. 293A and HCT116 were grown in standard conditions (10% FCS) except when assayed for SREBP (FCS was substituted for lipoprotein-deficient serum, Sigma). Aphidicolin (5µg/ml), nocodazole (40ng/ml) and bortezomib (0.5 µM) were used overnight. Cycloheximide was used at 50

µg/ml. Transfections were performed by calcium-phosphate precipitation.

Cell lysis, immunoprecipitations, immunoblotting, kinase assays. Protein extracts were made with NP-40 or Tween 20 lysis buffer, and SDS-PAGE, western blot, and immunoprecipitation utilized standard methods (Welcker et al. 2003). Quantitative western blotting was performed with an ImageQuant

Bioanalyzer and ImageQuant TL (Fig. 3D). Endogenous Fbw7 and SREBP were immunoprecipitated prior to detection by western blot.

SREBP1 pull-down. Bacterial recombinant 6xHis-tagged wild-type and dimer- deficient (mutation of three Leu residues in the zipper) SREBP1a (aa 1-490) were purified with Ni2+-NTA and phosphorylated with recombinant GSK3 (NEB) using 1mM ATP. Phosphorylated SREBP was monitored with pT456 (pT426 in

SREBP1b) antibody and incubated with GST-purified GST-Fbw7 expressed in

293 cells on beads.

Generation of dimer-deficient HCT116 cell-lines. Adeno-associated viral (AAV) vectors were used to target both endogenous Fbw7 alleles as described (Hirata and Russell 2000; Grim et al. 2008). An internal promoter-driven Neo vector was used to create Fbw7∆ D cells and a splice-acceptor-Neo vector used to develop additional lines with EWLKMF deletions (termed A7 and B6 in Fig. 3B).

Homozygous deletions were obtained by removing Neo from the first targeted allele with Ad-Cre, followed by retargeting and a second round of Cre-mediated excision. Homozygous mutants were identified by PCR, Southern blotting, and sequencing of genomic DNA and cDNA (SF4 and not shown).

Pulse-chase. Cells were starved for 30 min in methionine-free DMEM containing

5% dialyzed FCS and labeled in 6 cm dishes with 1 ml of trans-35S-label (300 µCi/ml) for 30 min. Dishes were washed, ‘chased’ with cold methionine, and frozen at -800C. Cell lysates were subject to immunoprecipitation and electrophoresis. Gels were analyzed with a phosphor-imager and quantified with

ImageQuant TL.

Ubiquitylation. Ubiquitylation reactions contained purified E1 enzyme (1 µg),

UbcH3 E2 enzyme (1 µg), neddylated Cul1/Rbx1 (100 ng), ubiquitin (5 µg), 2 mM

ATP, 5mM MgCl2, 2 mM DTT, 50 mM Tris pH 7.5. Cyclin E (Figs. 1F, SF1B, and

SF9) was added as transfected cell lysates (10 µl, spiked with bortezomib) whereas Fbw7 (25 ng) was added as purified recombinant monomer (aa 263-

707) or dimer (aa 228-707). Recombinant Fbw7 dimers were prepared as described (Pierce et al. 2013). For auto-ubiquitylation, purified Fbw7 was used as substrate, either as washed IPs (SF8C) or recombinant proteins (all other assays). The Fbw7 monomer was purified as described in Hao et al., 2007.

Supplemental references

Grim JE, Gustafson MP, Hirata RK, Hagar AC, Swanger J, Welcker M, Hwang HC, Ericsson J, Russell DW, Clurman BE. 2008. Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase. J Cell Biol 181: 913-920.

Hirata RK, Russell DW. 2000. Design and packaging of adeno-associated virus gene targeting vectors. J Virol 74: 4612-4620.

Pierce NW, Lee JE, Liu X, Sweredoski MJ, Graham RL, Larimore EA, Rome M, Zheng N, Clurman BE, Hess S et al. 2013. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153: 206-215. Welcker M, Singer J, Loeb KR, Grim J, Bloecher A, Gurien-West M, Clurman BE, Roberts JM. 2003. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation. Molecular cell 12: 381-392.