Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation

Abstract

GSK-3beta-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3beta could inhibit nuclear import. Here, we report that GSK-3beta-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3beta, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division.

Figure 1

Nuclear export of cyclin D1 during S phase. (A) NIH-3T3 cells were arrested in G₀ by serum deprivation for 36 h and subsequently stimulated to synchronously re-enter the cell cycle by addition of FCS. Cells were harvested and processed for immunofluorescence microscopy at 8 h (G1-phase, panels a, b) or 16 h (S-phase, panels c–h). Cyclin D1 was visualized with a cyclin D1–specific monoclonal antibody, and DNA was visualized with Hoechst dye. In certain experiments, 10 ng/mL LMB (panels e, f) or 20 mM LiCl (panels g, h) was added to culture medium before harvesting for immunofluorescence. (B) Lysates prepared from D1-3T3 cells left untreated or treated with 20 mM LiCl for 2 h or 10 ng/mL LMB for 2.5 h were precipitated (IP) with normal rabbit serum (NRS) or the M2 monoclonal antibody. Proteins were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membrane, and blotted with an antibody that specifically recognizes phosphorylated Thr-286. The membrane was subsequently stripped and reblotted with an antibody that recognizes both phosphorylated and unphosphorylated cyclin D1; then it was stripped once again and blotted with an antibody that recognizes CDK4. Sites of antibody binding were visualized by enhanced chemiluminescence.

Figure 2

GSK-3β promotes cyclin D1 nuclear export. (A) Asynchronous NIH-3T3 cells transiently overexpressing Flag-D1 and CDK4 (panels a, b), Flag-D1, CDK4, and wtGSK-3β (panels c–f), or Flag-D1, CDK4, and kinase dead (kd)GSK-3β (panels g, h) were harvested and processed for immunofluorescence. Cyclin D1 was visualized with the M2 monoclonal antibody, and cellular DNA was stained with Hoechst dye. In panels e and f, LMB was added to cellular medium at a final concentration of 10 ng/mL for 2.5 h before harvest. (B) Cyclin D1 localization in cells transfected and stained as in A was scored as either exclusively nuclear (e.g., panels a, e, g) or cytoplasmic (panel c). The number of cells containing nuclear cyclin D1, from four independent transfections, were counted and are shown graphically. The bars represent standard deviation between independent transfections.

Figure 3

Phosphorylation of Thr-286 facilitates cyclin D1 nuclear shuttling. (A) NIH-3T3 cells proliferating on glass coverslips and transiently expressing nucleoplasmin, NPc-M9 (panels a–c), cyclin D1 and CDK4 without (panels d–f) or with LMB (panels g–i), or D1-T286A and CDK4 (panels j–l) were treated with cycloheximide and fused with HeLa cells with polyethylene glycol. Thirty minutes after fusion, cells were harvested and processed for immunofluorescence microscopy. NPc-M9 was visualized using the 9E10 monoclonal antibody, and cyclin D1 was visualized with either a monoclonal antibody specific for murine cyclin D1 or the M2 monoclonal antibody, and DNA was visualized with Hoechst dye. The differential staining of human (homogenous) versus murine nuclei (punctate) by Hoechst dye 33258 facilitated the identification of fusions composed of murine and human nuclei. (B) The number of fusions wherein NPc-M9 and cyclin D1 were observed in HeLa nuclei was quantitated. The capacity of wild-type cyclin D1 and D1-T286A to shuttle in this assay in the presence of either LMB or LiCl was also determined. Frequencies were determined from multiple experiments and are expressed as the shuttling frequency relative to the positive shuttling control, NPc-M9.

Figure 4

CRM1 promotes the cytoplasmic localization of cyclin D1. (A) NIH-3T3 cells were transfected with plasmids encoding either wild-type cyclin D1 and CDK4 (panels a, b), cyclin D1, CDK4, and HA-CRM1 (panels c–e), D1-T286A and CDK4 (panels f, g), or D1-T286A, CDK4, and HA-CRM1 (panels h–j). Cyclin D1 was visualized with the anti-Flag octa-probe antibody (top), HA-tagged CRM1 was visualized with the 12CA5 mouse monoclonal antibody (center), and DNA was visualized with Hoechst dye (bottom). (B) Cyclin D1 localization in cells transfected and stained as in A, was scored as either exclusively nuclear (e.g., panels a, f, h) or cytoplasmic (panel c). The number of cells containing nuclear cyclin D1, from four independent transfections, were counted and are shown graphically. The bars represent standard deviation between independent transfections.

Figure 5

Thr-286 phosphorylation promotes cyclin D1-CRM1 binding. (A) Purified Flag-D1 phosphorylated with recombinant GSK-3β (lanes 1, 3), Flag-D1 treated with calf intestinal alkaline phosphatase (lane 2), or Flag-D1-T286A (lane 4) was separated on denaturing polyacrylamide gels and blotted with an antibody that specifically recognizes phosphorylated Thr-286 of cyclin D1. The membrane was subsequently stripped and reblotted with the cyclin D1 monoclonal antibody, which recognizes total cyclin D1. Sites of antibody binding were visualized by enhanced chemiluminescence. (B) 12CA5 Sepharose (lane 1) or recombinant HA-CRM1 bound to 12CA5 Sepharose was mixed with phosphorylated (D1-P), dephosphorylated cyclin D1 (D1), or cyclin D1-T286A in the absence or presence of Ran-GTP (lanes 2–7). After extensive washes with detergent-containing buffer, HA-CRM1-cyclin D1 complexes were denatured, resolved on polyacrylamide gels, and blotted with either the 12CA5 antibody for HA-CRM1 or the cyclin D1 monoclonal antibody. CRM1-associated D1 was quantitated by densitometry and is expressed relative to D1 binding to CRM1 in the absence of Ran-GTP. The precipitating antibody (α-HA) is indicated at bottom (IP). Sites of antibody binding were visualized by enhanced chemiluminescence.

Figure 6

Cyclin D1-T286A promotes cellular transformation. (A) Early (p4) or late (p19) passage cell lines were plated in complete medium containing 5% FCS at 4 × 10⁵ cells per 60-mm dish. Cells were grown for an additional 21 d, and foci were counted after staining with Giemsa. (B) NIH-3T3 (p22), D1-3T3 (p22), and D1-T286A-3T3 (p23) were plated in semisolid medium and cultured in a humidified environment for 21 d. Colonies were visualized by phase-contrast microscopy. Colonies >50 cells were scored. (C) Whole-cell lysates were prepared from cell lines established from D1-T286A-derived soft agar colonies or prepared from tumors that arose in SCID mice injected with either p9 or p23 D1-T286A-3T3 cells were precipitated with the cyclin D1 monoclonal antibody (lanes 1–6) and subjected to direct Western blotting with the cyclin D1 monoclonal antibody. Flag-D1 expressed in Sf9 cells served as a positive Western blotting control (lane 7). Sites of antibody binding were visualized by enhanced chemiluminescence. Relative levels of expression were determined by densitometry and are noted below the gel. (D) D1-T286A-3T3 cells or cells isolated from a D1-T286A-derived tumor (T23-3) were synchronized by serum deprivation and contact inhibition for 36 h and subsequently stimulated to synchronously re-enter the cell cycle by addition of FCS. Cells were harvested and processed for immunofluorescence microscopy at 8 h (G1 phase) or 16 h (S phase). Cyclin D1 was visualized with the cyclin D1–specific monoclonal antibody, and DNA was visualized with Hoechst dye.

Figure 7

Cellular transformation correlates with constitutive nuclear accumulation of cyclin D1-T286A. (A) Whole-cell lysates prepared from early (p11) or late (p18) NIH-3T3 or derivatives overexpressing either wild-type cyclin D1 or D1-T286A were subjected to direct Western blotting with the cyclin D1 monoclonal antibody. Sites of antibody binding were visualized by enhanced chemiluminescence. (B) Lysates were prepared as above and precipitated with either a control antibody (NRS) or the cyclin D1 monoclonal antibody. Precipitates were then assayed for their ability to phosphorylate recombinant GST-Rb. After SDS-PAGE, phosphorylated GST-Rb was visualized by autoradiography. (C) D1-3T3 (lanes 1–4) or D1-T286A-3T3 (lanes 5–7) cells were pulse-labeled with [³⁵S]methionine for the indicated intervals. Cyclin D1 was immunoprecipitated from lysates, resolved on a denaturing gel, and visualized by autoradiography. Accumulation of newly synthesized cyclin D1 protein was quantitated by densitometry (bottom). (D) Asynchronously proliferating early and late passages of the indicated cell lines were harvested and stained with propidium iodide; DNA content was determined by flow cytometry.