Cdk2-dependent Phosphorylation and Functional Inactivation of the pRB-related p130 Protein in pRB(−), p16INK4A(+) Tumor Cells
2000; Elsevier BV; Volume: 275; Issue: 39 Linguagem: Inglês
10.1074/jbc.m005707200
ISSN1083-351X
AutoresLengya Cheng, Ferdinando Rossi, Weizhao Fang, Takahiro Mori, David Cobrinik,
Tópico(s)Cancer Research and Treatments
ResumoThe retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G1 phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16INK4A in most pRB(−) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G1-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(−), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G1 and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(−), p16(+) cells as compared with pRB(+), p16(−) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins. The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G1 phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16INK4A in most pRB(−) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G1-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(−), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G1 and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(−), p16(+) cells as compared with pRB(+), p16(−) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins. cyclin-dependent kinase phosphate-buffered saline chloramphenicol acetyltransferase dominant negative In mammalian cells, proliferative decisions are frequently made in the late G1 phase of the cell cycle, when under appropriate conditions cells progress through the restriction point and become committed to proceed through the S, G2, and M cell cycle phases (1Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1905) Google Scholar, 2Planas-Silva M.D. Weinberg R.A. Curr. Opin. Cell Biol. 1997; 9: 768-772Crossref PubMed Scopus (209) Google Scholar). 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MDA-MB468, MDA-MB436, BT549, MDA-MB231, MDA-MB435S, ZR75–1, BT483, BT474, and HS578T cells were obtained from American Type Culture Collection and cultured under American Type Culture Collection-specified conditions. Subconfluent Saos-2 and HaCaT cells were trypsinized and plated at 3 × 106/100-mm dish and 1.5 × 106/100-mm dish, respectively. On the following day, cells were rinsed once with phosphate-buffered saline (PBS) and maintained in serum-free medium for 3 days. Cells were restimulated by addition of complete medium, in some cases in the presence of 1 mm hydroxyurea. Cells were seeded to 24-well plates at 2.5 × 104 cells/well for SAOS-2 or 6 × 104cells in 0.5 ml of medium/well for HaCaT, serum starved as above, and restimulated in the presence of 10 μCi/ml, [3H]thymidine for 1 h. Labeling was terminated by adding ascorbic acid to 250 mm, cells were washed three times with PBS and once with ice-cold 5% trichloroacetic acid, incubated in 5% trichloroacetic acid on ice for 30 min, washed with H2O, and lysed overnight in 1 m NaOH at 37 °C, and [3H]thymidine incorporation was measured by scintillation counting. Samples were assayed in triplicate, and the means and standard errors of [3H]thymidine incorporation levels were calculated. Flow cytometry was performed as described previously (15Zhu L. van den Heuvel S. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (477) Google Scholar). Cells were washed with ice-cold PBS, incubated in 1 ml of ice-cold cell lysis buffer (50 mm HEPES, 150 mm NaCl, 2.5 mm EGTA, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Tween 20, 10% glycerol, 10 mm β-glycerophosphate, 1 mm NaF, 0.1 mm Na3VO4, pH 7.5, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 25 μg/ml benzamidine, 10 μg/ml pepstatin A, and 2 mm phenylmethylsulfonyl fluoride for Fig. 4; and as described (15Zhu L. van den Heuvel S. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (477) Google Scholar) for Fig. 8 A), removed from plates by scraping, sonicated three times on ice for 10 s each at setting 3 on a Tekmar TM50 sonic disrupter, and centrifuged in a microcentrifuge at 14,000 rpm for 10 min at 4 °C. The supernatants were collected, and the protein concentrations were determined by a Coomassie Blue binding assay (Bio-Rad).Figure 8DNCdk2 impairs dissociation of p130 from E2F-4 and inhibits E2F-4-dependent transactivation. A, Saos-2 cells co-transfected with pBABE-Puro and expression vectors for either DNCdk2 or an empty vector control were serum starved and selected in the presence of puromycin and were then restimulated for 21 h in the presence of puromycin and hydroxyurea. Lysates (2 mg) were immunoprecipitated with anti-E2F-4 antibody and subjected to Western blotting, and the upper portion of the blot was probed sequentially with anti-p130 and anti-p107 antibodies, and the lower portion was probed with anti-E2F-4. B, Saos-2 cells were co-transfected with E2-CAT, CMV-lacZ, and expression vectors for E2F-4 + DP-1, DNCdk2, or the empty expression vector control, as indicated. CAT and β-galactosidase activity were measured 48 h after transfection, and CAT activity normalized to β-galactosidase was determined. Error bars indicate standard deviation for duplicate transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lysates were separated by electrophoresis through SDS-polyacrylamide gels, transferred to nitrocellulose, incubated in 0.05% Tween 20 in PBS (0.05% TPBS) containing 5% nonfat dry milk for 1 h, and washed three times for 10 min each in 0.05% TPBS. Membranes were probed overnight with shaking at 4 °C in 0.05% TPBS containing 2.5% nonfat dry milk and the indicated primary antibodies and then incubated with horseradish peroxidase-conjugated protein A/G (Pierce) at 1:1000 dilution or horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Pharmacia Biotech) at 1:3000 dilution for 1 h, and detected with enhanced chemiluminescence (Kirkegaard and Perry Laboratories). On occasion, membranes were stripped in 62.5 mm Tris, 0.7% β-mercaptoethanol, 2% SDS for 20 min at 50 °C and reprobed. The following antibodies and dilutions were used in Western analyses: polyclonal antibodies against Cdk2 (Santa Cruz SC-163 at 1:1000), Cdk3 (Santa Cruz SC-826 at 1:1000), Cdk4 (Santa Cruz SC-260 at 1:1000), Cdk6 (Santa Cruz SC-177 at 1:1000), p130 (Santa Cruz SC-317 at 1:1000, used for Figs. 2 A, 3, and 4), pRB (Santa Cruz SC-50 at 1:1000), p107 (Santa Cruz SC-318 at 1:1000), E2F-4 (Santa Cruz SC-866 at 1:200), cyclin E (Upstate Biotechnology 06-134 at 1:1000); monoclonal antibodies against p130 (Transduction LaboratoriesR27027/LI at 1:1000; used for Fig. 2 B, 7 A, and8 A), cyclin D1 (NeoMarkers MS-210 at 1:100), cyclin D2 (NeoMarkers MS-214 at 1:100) cyclin D3, (NeoMarkers MS-215 at 1:100), and p16 (NeoMarkers MS-218 at 1:100).Figure 3Absence of p130 Form III in pRB(−), p16(+) cells. Western blot analysis of p130, pRB, and p16 in exponentially growing mammary tumor cell lines is shown. p130 Form III was detected in each of the pRB(+), p16(−), but not in the pRB(−), p16(+) cell lines. Levels of p130 Form III as well as hyperphosphorylated pRB were lower in BT483 (lane 7) than in other pRB(+) cell types owing to the decreased growth rate of the culture.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Inhibition of p130 Form IIb production by dominant negative Cdk2. A, Saos-2 cells were co-transfected with the Hook gene and expression vectors for p16 (lanes 1 and 6), DNCdk2 (lanes 2 and 7), or DNCdk3 (lanes 3 and 4), or with empty expression vector (lanes 5 and 8) and were serum starved, restimulated for 21 h, and selected using magnetic beads. Protein lysates prepared from the selected (lanes 1–5) or unselected (lanes 6–8) cells were then subjected to Western blotting with anti-p130 antibody. The position of the p130 Form IIb is indicated. The impaired production of Form IIb in DNCdk2-expressing cells (lane 2) is most readily appreciated by comparing p130 mobility to that of a consistently migrating species that cross reacts with the p130 antibody (asterisk).B, the high level expression of p16 (lane 1), DNCdk2 (lane 2), and DNCdk3 (lane 4) relative to their endogenous counterparts in transfected and selected cells was confirmed by Western blot analysis using a mixture of the corresponding antibodies. C, the ability of DNCdk2 and DNCdk3 to induce a G1 block was confirmed by co-transfecting Saos-2 cells with an expression vector for CD20 together with vectors encoding either DNCdk2, DNCdk3, DNCdk4, or p16, as indicated, or with the “empty” cDNA3 vector control. At 48 h after transfection, cells were fixed, stained with fluorescein isothiocyanate-labeled anti-CD20 antibody and with propidium iodide, and DNA content of CD20(+) cells was determined by fluorescence-activated cell sorter analysis. The percent increase in G1 cells as compared with cDNA3 vector transfected controls was calculated. Error barsindicate standard deviation for duplicate transfections.View Large Image
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