Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line (original) (raw)

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Acknowledgements

We thank R. Klausner, F. Mercurio, M. Pagano, B. Sarcevic and Y. Xiong for providing reagents and D. von Seggern for help with adenovirus work. H.S. acknowledges support from the Breast Cancer Research Program (BCRP) of the University of California, and from the Breast Cancer Research Program of the US Department of Defense (DOD). C.S. is supported by a Special Fellow Award from the Leukemia and Lymphoma Society of America. P.K. is an APART fellow of the Austrian Academy of Sciences. This work was supported by a grant from the National Cancer Institute to S.I.R.

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Authors and Affiliations

1. Department of Molecular Biology, MB-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA
   Heimo Strohmaier, Charles H. Spruck, Peter Kaiser, Kwang-Ai Won & Steven I. Reed
2. Department of Oncology/Pathology, CCK R8:03, Karolinska Hospital, Stockholm, 171 76, Sweden
   Olle Sangfelt

Authors

1. Heimo Strohmaier
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2. Charles H. Spruck
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3. Peter Kaiser
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4. Kwang-Ai Won
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5. Olle Sangfelt
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6. Steven I. Reed
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Corresponding author

Correspondence toSteven I. Reed.

Supplementary information

Methods

Plasmids and baculovirus constructions

A human EST encoding part of the hCdc4 gene was amplified from HeLa mRNA by RT-PCR using two sequence-specific oligonucleotide primers, Pcr1 (5'-gcaagcttatgggtttctacggcacatt-3', forward), and Pcr2 (5'-atgggccctgctcttcacttcatgtcc-3', reverse), and TA-cloned into pCR2.1 (Invitrogen). The sequence of the cloned cDNA was verified in its entire length (1.7 Kb) by sequencing and found to match the sequence published in the NCBI database (Genbank accession no: BAA91986.1). The complete coding region corresponding to this cDNA was determined by 5’ RACE. Probing a Northern blot with sequences corresponding to specific exons, it was found that this cDNA corresponds to a 4 kb hCdc4 mRNA expressed at high levels in only some cell types. A second cDNA was identified by scanning upstream genomic sequences for probable exons and using these exons to probe a Northern blot. This cDNA was found to correspond to a ubiquitous 5.5 kb hCdc4 mRNA. Human cDNAs encoding the two full-length isoforms of the hCdc4 protein were amplified from HeLa mRNA by RT-PCR using sequence-specific oligonucleotide primers, Pcr4 (5'-cttttggaaatgaatcaggaa-3', forward) and Pcr2 (5'-atgggccctgctcttcacttcatgtcc-3', reverse) for the cDNA encoding the 110 kDa isoform, and Pcr5 (5'-catgtatgtatgtgtgtcccg-3', forward) and Pcr2 for the cDNA encoding the 69 kDa isoform, and subsequently TA-cloned into pCR2.1 (Invitrogen). The sequences of the cloned cDNAs were verified in their entire lengths (2.2 kb, and 1.9 kb, respectively) by sequencing. The cDNA encoding the 110 kDa isoform was deposited in GenBank (accession number: AY049984). A mammalian transfection plasmid expressing N-terminal Flag-tagged hCdc4 protein was constructed by subcloning into pFLAG-CMV2 (Sigma). For expression in E. Coli, hCdc4 was tagged at the N-terminus with a RGS.His epitope through subcloning into pQE-10 (Qiagen). Complementary DNAs encoding hCdc4 and a DF-box-mutant that had been deleted for its F-box using a two-step PCR protocol, as well as the cDNA coding for b-galactosidase were cloned into pDV46. Recombinant adenoviruses were generated by co-transfecting the recombinant plasmids and pBHG1022 into 293 cells using the calcium phosphate precipitation method. The b-TrCP and Skp2 clones were gifts from Dr. F. Mercurio (Signal Pharmaceuticals, San Diego), and Dr. M. Pagano (Department of Pathology and Kaplan Comprehensive Cancer Center, New York University, New York), respectively, and were cloned into pFLAG-CMV2 to obtain b-TrCP and Skp2 tagged at their N-termini with the Flag-epitope. The mammalian transfection plasmid pCDNA3-Cul1-HA was a gift from Dr. R. Klausner (NIH, Bethesda, Maryland), and pCDNA3-3MYCROC1 as well as pCDNA3-hSkp1 were gifts from Dr. Y. Xiong (Lineberger Cancer Center, University of North Carolina at Chapel Hill). Baculovirus expressing cyclin E with a GST-tag at its N-terminus was a gift from Dr. B. Sarcevic (The Garvan Institute, Sydney, Australia). Recombinant baculoviruses expressing GST-tagged versions of cyclin E phosphorylation site mutants (T62A, T380A, T62A/T380A) were generated using the pFastBac-system (Gibco BRL) according to the manufacturer's protocol. Baculovirus encoded proteins were expressed in SF9 insect cells grown in Ex-Cell 401 media (JRH) supplemented with 2% fetal bovine serum.

Analysis of cyclin E turnover in yeast

All yeast strains are isogenic to 15DaubD, a_bar1D_ ura3Dns, a derivative of BF264-15D23. Several temperature sensitive skp1 mutants with different cell cycle arrest phenotypes were constructed by a combination of PCR mutagenesis and in vivo gap-repair similar to the procedure described by Muhlrad et al.24. The mutant shown in Figure 1 (skp1-24) arrested with 1C DNA content and a multi-budded phenotype. To analyze cyclin E turnover in various yeast mutants expressing cyclin E from the inducible GAL1 promoter, cells were grown in YEP-raffinose at 25°C to an OD600= 0.3. Cells were then shifted to 35°C and after 30 min galactose was added to a final concentration of 2% to induce the GAL1 promoter. To terminate cyclin E expression after 60 min, cells were collected on filters and transferred to YEPD media and incubation was continued at 35°C. Extracts were prepared from aliquots taken after the periods indicated and analyzed for cyclin E by Western blotting using monoclonal anti-cyclin E antibodies (HE12). Cells were lysed in RIPA buffer (1% deoxycholic acid, 1% Triton-X-100, 0.1% SDS, 250 mM NaCl, 50 mM Tris-HCl pH 7.5). A similar protocol was used to analyze the turnover of cyclin E phosphorylation site mutants in yeast except that a strain was used where human CDK2 was substituted for the endogenous Cdk Cdc288.

Cell culture and immunological techniques

A panel of breast cancer-derived cell lines were obtained from ATCC and the University of Michigan Breast Cell/Tissue Bank and Database and grown in media recommended by the suppliers. HeLa, Kb (human epidermoid carcinoma), and 293T cells were grown in DMEM (Gibco BRL) supplemented with 10% fetal bovine serum. All cells were maintained in a humidified 37°C incubator with 5% CO2. 293T cells were transfected with various combinations of plasmids in 10 cm-dishes using the calcium phosphate precipitation method. Forty hours post-transfection, cells were lysed, as indicated, into either mammalian cell lysis buffer 1, MCLB1 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) or mammalian cell lysis buffer 2, MCLB2 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% NP-40, 10% glycerol, 1 mM EDTA, 1 mM DTT), supplemented with 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethyl sulfonylflouride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin. After centrifugation at 4°C (14.000 rpm, 15 min) lysates (1 mg/0.5 ml) were subjected to immunoprecipitation using 10 mg anti-Flag antibody immobilized on agarose beads (M2, Sigma) for 2 hours at 4°C. Immune complexes were washed three times with lysis buffer and subsequently used for ubiquitination reactions or analyzed by immunoblotting. Polyclonal antibodies against human Cdc4 were generated in rabbits after injection of recombinant Cdc4 protein produced in bacteria. Antibodies were affinity-purified using nitrocellulose-bound antigen. Commercially obtained antibodies used in this study: Mouse monoclonal anti-Flag antibody (M2, Sigma), mouse monoclonal anti-HA antibody (HA.11, Babco), rabbit polyclonal anti-Myc antibody (A-14, Santa Cruz), rabbit polyclonal anti-Cdk2 antibody (M2, Santa Cruz), and rabbit polyclonal antibodies against Cul1, Skp1, and ROC1 (Neomarkers).

Pulse-chase, Northern and Southern blot analysis

Pulse-chase experiments were performed on thymidine arrested cells as described25. Thymidine arrested Kb cells were co-transduced with adenovirus encoding cyclin E (resulting in a 5-10 fold elevation over endogenous levels) and virus encoding either wild-type hCdc4, an F-box deleted hCdc4, or control b-galactosidase. Viral transductions were incubated for 24 hours prior to pulse-chase. Immunoprecipitations were performed with a monoclonal anti-cyclin E antibody (HE172). Quantitation of bands was performed using ImageQuant software (Molecular Dynamics). For Northern blot analysis, 2 mg of poly[A]+ RNA was isolated from asynchronously growing cultures according to the manufacturer's protocol (Qiagen) and run on a 1% formaldehyde agarose gel as described26. The gel was blotted onto Zeta-Probe GT genomic membrane (Bio-Rad) and hybridized with a radiolabeled hCdc4 probe followed by autoradiography. For Southern blot analysis, 10;g of DNA was digested with _Sst_I and _Eco_RV, run on 0.8% agarose gels, blotted and probed with a genomic fragment corresponding to Exons 8 and 9.

In vitro binding

Complementary cDNAs encoding various hCdc4 isoforms and mutants, b-TrCP, and Skp2 were in vitro translated into 35S methionine-labeled proteins using a T7 transcription/translation system (Promega). GST-tagged cyclin E and various cyclin E phosphorylation site mutants were expressed in baculovirus infected SF9 insect cells and adsorbed on glutathione beads for 1 hour at 4°C after lysing the cells into GST-lysis buffer (50 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 0.5% Tween-20, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) supplemented with 5 mM sodium flouride, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate, 1 mM phenylmethyl sulfonylflouride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin. After washing the beads first with lysis buffer and then with wash buffer (50 mM HEPES-NaOH pH 7.5, 0.01% Tween-20, 10% glycerol, 1 mM EDTA, 1 mM DTT) they were subjected to either phosphorylation reaction in kinase assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2) in the presence of 1 mM ATP or dephosphorylation reaction using l–phosphatase (NEB) for 1 hour at 30°C. The beads were washed three times with binding buffer (20 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT) prior to incubation with in vitro translated [35S] methionine-labeled proteins in 200 ml binding buffer for 2 hours at 4°C. Bound proteins were analyzed by SDS-PAGE followed by autoradiography.

In vitro ubiquitination assay

Recombinant SCF complexes containing different Flag-tagged F-box proteins were isolated from transfected 293T cells lysed into mammalian cell lysis buffer 2 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% NP-40, 10% glycerol, 1 mM EDTA, 1 mM DTT), supplemented with 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethyl sulfonylflouride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin) using anti-Flag antibodies immobilized on agarose beads. Individual immune complexes were washed three times with lysis buffer and three times with ubiquitination reaction buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT). GST-tagged cyclin E and cyclin E phosphorylation site mutants purified from baculovirus infected SF9 insect cells on glutathione beads were subjected to either phosphorylation or dephosphorylation followed by elution of the bound proteins with elution buffer (50 mM HEPES-NaOH pH 7.5, 0.01% Tween-20, 10% glycerol, 1 mM EDTA, 1 mM DTT) containing 15 mM reduced glutathione. Eluates from multiple elution steps were pooled and concentrated on Centricon-30 spin columns (Amicon) and stored in aliquots at –80°C. Equal amounts of SCF immune complexes were mixed with different eluted cyclin E proteins for 30 min on ice to allow binding. Subsequently, aliquots of this mixture were added to ubiquitination reactions in a total volume of 30 ml containing 15 mg of bovine ubiquitin (Sigma), 0.5 mg of yeast E1 enzyme (Boston Biochem), 1 mg of human 6xHis-Cdc34 purified from bacteria, and an ATP-regenerating system (1 mM ATP, 20 mM creatine phosphate, 0.1 mg/ml creatine kinase) in ubiquitination reaction buffer25 supplemented with 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethyl sulfonylflouride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin. The CDK2-inhibitor roscovitine (Biomol; 100 mM final concentration) was added to reactions containing dephosphorylated cyclin E as substrate. Reactions were incubated at 30°C for 2 hours, terminated by boiling for 5 min with SDS-sample buffer, and analyzed by SDS-PAGE followed by immunoblotting using anti-cyclin E antibodies. The ubiquitination assay using p27 as a substrate was performed as described25.

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Strohmaier, H., Spruck, C., Kaiser, P. et al. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.Nature 413, 316–322 (2001). https://doi.org/10.1038/35095076

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• Received: 17 April 2001
• Accepted: 28 August 2001
• Issue Date: 20 September 2001
• DOI: https://doi.org/10.1038/35095076