Lysine 269 is essential for cyclin D1 ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ligase and subsequent proteasome-dependent degradation

Abstract

Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, through Gly-76 of ubiquitin, to an varepsilonNH2 group of an internal lysine residue in a given substrate. Although E3 ligases frequently use lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome through the polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid polyubiquitin-mediated degradation of cyclin D1. Mutation of Lys-269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1 but also triggers cyclin D1 accumulation within the nucleus, thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress-induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in response to DNA-damaging agents. Strikingly, although mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo, showing that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome.

Figure 1. Mutation of Lys-269 in cyclin D1 leads to increased cyclin D1 protein stability without the attenuation of cyclin D1 functions

A. NIH-3T3 cells stably expressing wild-type cyclin D1, D1-K238R and D1-K269R were treated with 100μg/ml cyclohexemide for indicated periods of time. Cells were harvested and cyclin D1 protein levels were determined by immunoblot with D1-72-13G antibody. B. NIH 3T3 cells expressing wild type cyclin D1, D1-T286A, or D1-K269R were irradiated and harvested as indicated. Cyclin D1 levels were assessed by immunoblot. C. Cell lysates from NIH-3T3 cell lines stably expressing WT cyclin D1, D1-T286A and D1-K269R were used for immunoprecipitation of cyclin D1 with M2 agarose followed by immunoblot with phospho-Thr-286, D1-72-13G and cdk4 antibodies. D. Same as B, immunoblot with p27 antibody. E. Cyclin D1/cdk4 complexes were assembled in SF9 cells and used to phosphorylate recombinant GST-Rb, followed by autoradiography.

Figure 2. D1-K269R is resistant to SCF^{Fbx4/αB-crystallin} ubiquitylation in vitro

A. SCF^{Fbx4/αB-crystallin} complexes were assembled in SF9 cells, purified with M2 agarose and used to ubiquitylate recombinant GST-D1 or GST-D1-K269R. Reaction mixtures were separated by SDS-PAGE, followed by immunoblot with cyclin D1 antibody. B. D1/K4 and D1-K269R/K4 complexes were purified form SF9 cells using M2 agarose and used for in vitro ubiquitylation reaction as described in A. C. SCF^{Fbx4/αB-crystallin} complexes were expressed in SF9 cells, purified with M2 agarose and mixed with GST-WT cyclin D1, D1-T286A or D1-K269R for binding analysis. Complexes were separated by SDS-PAGE, followed by immunoblot with cyclin D1 and Fbx4 antibodies.

Figure 3. In vivo ubiquitylation of D1-K269R

A. NIH-3T3 cells stably expressing WT and K269R cyclin D1 were treated with 10μM MG132 for 6 hours, proteins were lysed under denaturing conditions (50mM Tris-HCl(pH 7.4), 1% SDS and 5mM DTT). Proteins were precipitated with anti-ubiquitin antibody in the buffer containing 50mM Tris-HCL (pH 7.4), 250mM NaCl, 5mM EDTA and 0.5% NP-40 and separated by SDS-PAGE and detected by immunoblot with a cyclin D1 antibody. B. NIH-3T3 cells stably expressing WT cyclin D1, D1-T286A and D1-K269R cyclin D1 were transfected with WT, K48R, K0 and K63R ubiquitin constructs. 48 hours post-transfection cells were treated with 10 μM MG132 for 6 hours and cell lysates were immunoprecipitated with M2 agarose followed by SDS-PAGE of protein complexes and immunoblot with anti-HA, cyclin D1 and Fbx4 antibodies. C. NIH-3T3 stably expressing D1-K269R were transfected with an Fbx4 expression vector. 48 hours post-transfections cells were used for cycloheximide chase assay at indicated time intervals. Cell lysates were separated by SDS-PAGE, followed by immunoblot with cyclin D1, Fbx4 and p27 antibodies. D. 293T cells were transfected with ubiquitin, ΔF-Fbx4 and cyclin D1 constructs as indicated. 48 hours post-transfection cells were treated with 10 μM MG132 for 6 hours and cell lysates were immunoprecipitated with anti-cyclin D1 antibody followed by SDS-PAGE of protein complexes and immunoblot with the cyclin D1 antibody. E. Same as in B, followed by immunoblot with cyclin D1 and PSMD7 antibodies.

Figure 4. D1-K269R has increased nuclear localization

A. NIH-3T3 cells stably expressing WT cyclin D1, D1-K269R and D1-T286A were subjected to immunofluorescence analysis with cyclin D1 antibody (green), slides were counterstained with DAPI (blue). B. Quantification of A from three independent experiments (error bars represent standard deviation). C. Cell cycle analysis of asynchronous NIH-3T3 cell lines by PI/FACS.

Figure 5. K269R cyclin D1 transforms NIH-3T3 cells

A. Anchorage –independent growth of NIH-3T3 cells stably expressing WT cyclin D1, D1-T286A, D1-K269R or pBabe-puro vector was analyzed by growth in soft agar. Colonies were visualized by microscopy. B. Quantification of triplicate samples shown in A. C. Foci formation ability of NIH-3T3 cells stably expressing WT cyclin D1, D1-T286A and D1-K269R was determined by Giemsa stain of foci grown for 21 days.