PMC

GSK-3β overexpression redirects cyclin D1 to the cytoplasm. (A) NIH-3T3 cells transiently expressing CDK4 and either Flag-tagged cyclin D1 (left), Flag-tagged cyclin D1 plus wild-type GSK-3β (wtGSK–3β; middle), or Flag-tagged cyclin D1 plus kinase-defective GSK-3β (kdGSK-3β; right) were fixed and processed for immunofluorescence. Flag-tagged cyclin D1 was visualized by staining with the M2 monoclonal antibody to the tag (top), and cellular DNA was stained with Hoechst dye (bottom). (B) The subcellular localization of Flag-tagged cyclin D1 or Flag-tagged D1-(T286A) without ectopic GSK-3β or with either wtGSK-3β or kdGSK-3β was determined by immunofluorescent staining with the M2 monoclonal antibody as above. The percentage of cells expressing exclusively cytoplasmic cyclin D1 or D1-(T286A) is presented graphically and represents the average of at least four independent experiments. Vertical bars indicate standard deviations from the mean.

Overexpression of MyrAkt stabilizes cyclin D1 in vivo. (A) NIH-3T3 cells infected with virus encoding the T-cell coreceptor (CD8; lanes 1–6) or MyrAkt (lanes 7–11) were labeled with [³⁵S]methionine for 30 min and then chased in the presence of excess unlabeled precursor for the indicated times. Radiolabeled cyclin D1 was precipitated with antibody to cyclin D1 and resolved on denaturing polyacrylamide gels (autoradiographic exposure time, 16 hr). The position of labeled cyclin D1 is indicated. (B) NIH-3T3 cells infected with virus encoding CD8 (lane 1) or MyrAkt (lanes 2,3) were labeled with [³⁵S]methionine. Detergent lysates were subjected to precipitation with either NRS (lane 3) or antiserum specific for a carboxy-terminal epitope of c-Akt (lanes 1,2). Immune complexes were resolved on a denaturing polyacrylamide gel, and MyrAkt was visualized by autoradiography (exposure time 16 hr).

Depletion of GSK-3β from mammalian cell lysates abrogates phosphorylation of cyclin D1 on Thr-286. (A) Detergent lysates prepared from NIH-3T3 cells were precipitated with either NRS (lane 1) or antibody to GSK-3β (lanes 2,3). Immune complexes were tested for their ability to phosphorylate GST-D1 (lanes 1,2) or GST–D1-(T286A) (lane 3) containing the carboxy-terminal 41 amino acids of cyclin D1. Phosphorylated GST-fusion proteins were resolved on polyacrylamide gels (exposure time 12 hr). (B) NIH-3T3 cell lysates were subjected to two rounds of depletion with either a control antibody, 9E10 (lanes 1,2), or with antibody to GSK-3β (lanes 3,4). Depleted lysates were mixed with [γ-³²P]ATP in kinase reactions performed with GST–D1 (lanes 1,3) or GST–D1-(T286A) (lanes 2, 4) prephosphorylated by PKA. Radiolabeled GST–D1 proteins were collected on glutathione–Sepharose beads and resolved on a denaturing polyacrylamide gel (autoradiographic exposure time 12 hr).

Subcellular localization of GSK-3β throughout the cell cycle. (A) NIH-3T3 cells engineered to overexpress Flag-tagged wild-type cyclin D1 were arrested by serum deprivation and contact inhibition and then dispersed and restimulated with serum to synchronously enter the cell cycle. Cells plated on glass coverslips in complete medium were fixed for immunofluorecence at the indicated times after serum addition, and were stained by use of a monoclonal antibody directed to GSK-3β (top). Cellular DNA was visualized with Hoechst dye (bottom). (B) At 6 hr (G₁ phase) and 16 hr (S phase) after cell cycle entry, cells from parallel cultures were disrupted, and equal quantitites of protein from nuclear and cytoplasmic fractions were separated on denaturing gels, transferred to membrane, and blotted with antibody to GSK-3β. Sites of antibody binding were detected by enhanced chemiluminescence.

GSK-3β phosphorylates cyclin D1 on Thr-286. (A) Cyclin D1 (odd lanes) or cyclin D1-(T286A) (even lanes) immunoprecipitated from Sf9 cells infected with baculoviruses encoding D1 and CDK4 were mixed with recombinant GSK-3β (lanes 1,2), ERK2 (lanes 3,4), SAPK (lanes 5,6), or cyclin E–CDK2 (lanes 7,8) plus [γ-³²P]ATP. After incubation at 30°C for 30 min, phosphorylated proteins were separated on a denaturing polyacrylamide gel, transferred to an Immobilon-P membrane, and visualized by autoradiography. The position of phosphorylated cyclin D1 is indicated. (B) Membrane slices containing cyclin D1 phosphorylated by GSK-3β in vitro (left), phosphorylated by endogenous Sf9 kinases (middle), or by a mixture of the two (right) were digested with trypsin and separated sequentially by electrophoresis and ascending chromatography. Phosphopeptides were visualized by autoradiographic exposure for 48 hr. The phosphopeptide containing Thr-286 was previously designated peptide A. Serine-containing peptides phosphorylated at lower stoichiometry can only be visualized after longer exposures (; ).

Cell cycle-dependent redistribution of cyclin D1 depends on the integrity of Thr-286. NIH-3T3 cells engineered to overexpress wild-type, Flag-tagged cyclin D1 (A) or Flag-tagged cyclin D1-(T286A) (B) were arrested in G₀ by serum deprivation and contact inhibition and then stimulated to synchronously enter the cell cycle. Cells were fixed at the indicated times after serum addition. The subcellular localization of D1 proteins was determined by staining with the cyclin D1 monoclonal antibody (top), and total cellular DNA was visualized with Hoechst dye (middle). In parallel, cells released from G₀ were stimulated to enter the cell cycle in the presence of BrdU to monitor S-phase entry. The percentage of cells incorporating BrdU at each time point is indicated below the panels. Because the levels of ectopic D1 expression were four- to eightfold above the endogenous background, a contribution of endogenous D1 to the staining pattern was negligible at the exposure chosen.

GSK-3β binds to D1 subunits and preferentially phosphorylates cyclin D1 in complexes with CDK4. (A) Sf9 cells infected with baculoviruses encoding cyclin D1 or cyclin D1 plus CDK4 with or without wild-type GSK-3β as indicated were metabolically labeled with [³²P]orthophosphate. Lysates were subjected to precipitation with NRS (lane 1), antibody to cyclin D1 (lanes 2,3), or antiserum specific for the carboxyl terminus of CDK4 (lanes 4,5) as indicated. Phosphorylated proteins were resolved on denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. Following autoradiography, the membrane was blotted with the antibody to cyclin D1 (bottom) and sites of antibody binding were visualized by enhanced chemiluminescence. (B) Sf9 cells infected with baculoviruses encoding cyclin D1 and CDK4 together with wild-type (wt) or kinase-defective (kd) GSK-3β were labeled with [³²P]orthophosphate. Lysates were subjected to precipitation with NRS or anti-D1 as indicated, and phosphorylated proteins were resolved on a denaturing polyacrylamide gel followed by transfer to Immobilon-P membrane (autoradiographic exposure time 12 hr; top). Following autoradiography, the membrane was blotted with the antibody to cyclin D1 as in A. The relative ratio of ³²P-labeled cyclin D1 versus total cyclin D1 in each lysate (densitometric scanning) is indicated between the two autoradiographs. (C) Sf9 lysates infected with baculoviruses encoding the proteins indicated were precipitated with the indicated antibodies and blotted with a monoclonal antibody to GSK-3β. Sites of antibody binding were visualized by enhanced chemiluminesence.