pEBB-Abl WT and pSG5-Abl KD (expressing the kinase-dead mutant K290R) were supplied by Ricardo Sanchez-Prieto, pCMV-N-Myc, pCMV-HA-p300, pCDNA3-HA-Pin1 WT and Y23A were supplied by Gianni del Sal, the Bcr-Abl plasmids pKI-210 WT and pKI 210 KD (expressing the kinase-dead mutant) were supplied by Warren Pear and pCEFL-GFPAbl by Silvio Gutkind. pcDNA-210 was supplied by Dr Pier Giuseppe Pelicci. For the mapping of interaction and phosphorylation sites on Myc we used plasmids pCβF-Myc Flag, pCβF-MycΔ1-110 Flag, pCβF-MycΔ1-180 Flag, kindly provided by M. Cole, as well as pCDNA3-Myc (1-262)-HA (mutant C in ; our ref. number BA1594), pCDNA3-Myc(1-347)-HA (mutant B; BA1595), pCDNA3-Myc(1-145)-HA (mutant C; BA1593), pCDNA3-Myc(263-439)-HA (mutant A; BA1518), pCMV-Myc-T58A-Flag (BA1987), pCMV-Myc-S62A-Flag (BA1986), pCMV-Myc-T58A-S62A-Flag (BA2235) and pGEX-Myc 1-262 (BA91). The Myc deletion mutant Δ1-24 and point mutants thereof were generated using site-directed mutagenesis by PCR, subcloning in a pCMV Myc-Flag vector, and confirmation by DNA sequencing. The constructs were pCMV-Δ1-24 Myc Flag (mutant G; BA2029), pCMV-Δ1-24 Y32F Myc Flag (mutant H; BA2030), pCMV-Δ1-24 Y74F Myc Flag (mutant I; BA2088), pCMV-Δ1-24 Y32-74F Myc Flag (mutant J; BA2089), pCMV-Myc Flag (BA2046), pCMV-Y12,16,22,32,74F Myc Flag (mutant 5YF; BA2095), pCMV-Y12,16,22,74F Myc Flag (BA2094), pCMV-Y12,16,22,32F Myc Flag (BA2093), pCMV-Y12,16,22F Myc Flag (BA2023), pCMV-Y32F Myc Flag (BA2090), pCMV-Y74F Myc Flag (BA2091). To generate these mutant constructs we performed PCR with the following primers:
PCR1: 5′primer (AP5435): GAAGAAGGATCCCCGGGCGAGC; 3′primer (AP5438): CGGCTGCACCGAGTCGWAGTCAAGGTCGWAGTTCCTGTTGGT
PCR2: 5′ primer (AP5437): GACTCGGTGCAGCCGTWCTTCTACTGCGAC 3′primer (AP5436 after the ClaI site in c-myc): AGCAGAAGGTGATCCAGACTCTGAC
PCR3: Used the combined PCR1 and PCR2 products as template, with primers AP5435 and AP5436
Myc Δ1-24 Y32F
PCR1: 5′primer (AP5441): GAAGAAGGATCCAACATGTGCGACGAGGAGGAGAACTTCTWCCAGCA; 3′primer (AP5436).
PCR1: 5′primer (AP5435): GAAGAAGGATCCCCGGGCGAGC; 3′primer (AP5568): GCTGCTGGAAGAAGTTCTCCTC
PCR2: 5′ primer (AP5567): GAACTTCTTCCAGCAGCAGCA; 3′primer (AP5436 after the ClaI site in c-myc): AGCAGAAGGTGATCCAGACTCTGAC
PCR3: 5′primer (AP5435); 3′primer (AP5436)
PCR1: 5′primer (AP5435) GAAGAAGGATCCCCGGGCGAGC; 3′primer (AP5570): GCAACGAAGGAGGGCGA
PCR2: 5′ primer (AP5569): CCTCCTTCGTTGCGGTCA; 3′primer (AP5436 after the ClaI site in c-myc): AGCAGAAGGTGATCCAGACTCTGAC
PCR3: 5′primer (AP5435); 3′primer (AP5436)
Fusion of 12/16/22 mutations and 32/74 mutations
PCR1: 5′primer (AP5435): GAAGAAGGATCCCCGGGCGAGC; 3′primer (AP6008): GAAGTTCTCCTCCTCGTCGCA
PCR2: 5′ primer (AP6007): TTCTACTGCGACGAGGAGGAGAAC; 3′primer (AP5436 after the ClaI site in c-myc): AGCAGAAGGTGATCCAGACTCTGAC
PCR3: 5′primer (AP5435); 3′primer (AP5436)
For Rat1 infection we used pBH2-HA-Flag-Myc (BA2313) and pBH2-HA-Flag-Myc Y74F (BA2315)
The following antibodies were used: anti Abl (K-19), anti Myc (C-33) anti Myc (N-262) and anti N-Myc (H-50) from Santa Cruz, Myc (Y69) from Abcam; anti HA from Covance (MMS-101P); anti Flag (F3165), anti-Flag beads (A2220) and anti-Vinculin (V9264) from Sigma; anti phosphotyrosine (4G10) and anti-Pin1 (PC270) from Upstate; anti acetyl lysine (#9441), anti P-Abl (Y412), and anti-Myc phospho-Thr 58/Ser 62 (#9401) from Cell Signaling.
The antibody directed against phospho-Tyr 74 in Myc (here anti Myc-pY74) was generated by Abcam upon our request and is commercially available (Abcam ab46848).
In order to selectively investigate the phosphorylation status of Myc Ser 62, rabbit polyclonal antisera were raised against a phosphorylated peptide containing phospho-Ser 62. For the immunization and subsequent affinity purification steps the following peptides were synthesized:
|- Myc ELL pS62:||ELLPTPPL(p)SPSRRSGLC|
|- Myc ELL pT58:||ELLP(p)TPPLSPSRRSGLC|
|- Myc ELL pT58-S62:||ELLP(p)TPPL(p)SPSRRSGLC|
|- Myc ELL:||ELLPTPPLSPSRRSGLC|
Crude bleeds were first immunodepleted with Myc ELL pT58, Myc ELL pT58-S62 and non-phosphorylated Myc ELL, and then affinity purified against the immunogenic peptide (Myc ELL p-S62). For characterization, the purified antibodies were used to immunoblot lysates from 293T cells transfected with expression vectors encoding either Flag-tagged Myc wild type or T58A/S62A mutants. As shown in figure S1
, purified anti-Myc p62 efficiently recognized both Myc WT and T58A while, as expected, no signal was detected for the S62A mutant (Fig. S10
Cell Culture, Transfections and Reagents
K562, MOLM, KYO, 293T, U2OS, and HeLa cells were growth under standard conditions. Pin1 KO MEFs were cultivated in DMEM medium supplemented with Glutamine 10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin, NEAA and β-mercaptoethanol. Cells were transfected using lipofectamine (Invitrogen) following the protocol supplied by the vendor. The Abl inhibitor STI571 was kindly provided by Novartis (Basel, Switzerland).
Immunoprecipitation, co-immunoprecipitation and In Vitro Binding
GST-Pin1 pull-down assays were performed as described (Zacchi el al 2002). Immunoprecipitation was carried out essentially as described in (Haupt et al 1996).
Immunofluorescence and Immunohistochemistry
For Immunofluorescence, adherent cells were grown on glass slides and fixed for 10 min in 4% paraformaldehyde. Cells growing in suspension were subjected to a cytospin (20g 5 min) and fixed as above. Cells were permeabilized with 0.2 % Triton in PBS (10 min) and blocked in 5% BSA (30 min, RT), followed by incubation with the primary antibody in 5% BSA (1h 30 min, RT), and with the secondary antibody (cy3 anti-rabbit) in 5% BSA (60 min, RT). The slides were finally stained with DAPI and mounted with Mowiol Mounting Media.
Immunohistochemical staining was performed with the avidin-biotin-peroxidase technique. Five- micrometer-thick sections were cut from the tissue specimens and placed on poly-L-lysine–coated glass slides. Sections were deparaffined by xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.1% hydrogen peroxidase in absolute methanol for 20 min. For antigen retrieval, the tissue sections were heated in a pressure cooker in citric acid monohydrate 10 mM, pH 9.0, for 5 min, and then incubated with the primary antibody at room temperature. IHC was performed with Benchmark XT (Ventana Medical Systems, Inc, Tucson, AZ). The primary antibodies and dilutions used were: anti-p-Abl (p-Y412 Cell Signalling Technology 1:50), anti-Myc-pY74 (Abcam ab46848 1:50), anti-Myc (Y69 rabbit monoclonal, Abcam ab32072, 1/100). The incubation time for the antibodies was 60, 120 and 32 min respectively. All slides were hematoxylin counterstained, dehydrated, and mounted. Negative controls were performed by omitting the primary antibody.
293T cells were transfected with Myc WT or 5YF mutant. Myc was immunoprecipitated using anti-Flag beads, followed by three washes in IP buffer (50mM Tris pH 8, 5mM EDTA, 150-300mM NaCl, 0.5% NP-40) and one wash in kinase buffer. Immunoprecipitates were resuspended in kinase buffer (60mM HEPES, 5mM MgCl2, 5mM MnCl2, 3mM Na2VO4, 1.25mM DTT, 200 mM ATP) adding 50ng Abl1 Kinase (Cell Signalling). The reaction was incubated at room temperature for 30min. Immunoprecipitates were analysed by immunoblot with the anti-phosphotyrosine 4G10 antibody.
Dual Luciferase assay
U2OS cells were transfected with Fugene with 200ng of pNuc-Luc reporter, 200ng of pCMV-Flag-Myc, 100ng pcDNA-HA-Pin1, 50ng pCMV-HA-p300. For normalization and transfection efficiency control we used 8ng of pRL-TK reporter (Promega) that constitutively expresses the Renilla luciferase. After 36 hours cells were lysed and assayed for luciferase activity using the Dual Luciferase kit (Promega).
Rat1 co-transformation assay
Co-transformation of Rat1 cells by Myc and Bcr-Abl was as previously described (19
). To produce recombinant retroviruses, Phoenix eco cells were transfected with retroviral constructs expressing Bcr-Abl (pKI-Bcr-Abl WT or KD) or Myc (pBabe-Hygro-Myc-Flag, WT or Y74F). Rat1 cells were infected with either form of the Bcr-Abl viruses, selected with Puromycin, super-infected with either form of the Myc viruses, and selected with Hygromycin. Infected cells were directly plated in triplicate in medium containing 0.3% Bacto-agar laid on top of a layer of 0.6% Bacto-agar, and cultured for 2 weeks. Fresh media was changed every 2 days. Colonies were visualized by 0.1% p-iodonitro tetrazolium violet (INT, Sigma) staining.