Extended Data Fig. 4 |. Mutations in the TP motif of D-type cyclins protect them from AMBRA1-mediated degradation.

a, Protein sequence alignment of the extreme C termini of D-type cyclins from different source organisms. Amino acids highlighted in red correspond to the highly conserved TP motif. Asterisks indicate positions that are fully conserved. Colons indicate conservation between groups of strongly similar properties. The dot indicates conservation between groups of weakly similar properties. The alignment was performed in UniProt (https://www.uniprot.org/) using the Clustal Omega algorithm. UniProt identifiers are shown on the left. Source organisms: HUMAN, H. sapiens; BOVIN, Bos taurus; CANLF, Canis familiaris; MOUSE, Mus musculus; RAT, Rattus norvegicus; DANRE, Danio rerio; BRAFL, Branchiostoma floridae; STRPU, Strongylocentrotus purpuratus; CRAGI, Crassostrea gigas; LOTGI, Lottia gigantean; OCTBM, Octopus bimaculoides; STRMM, Strigamia maritima; BOMMO, Bombyx mori; ATTCE, Atta cephalotes; APIME, Apis mellifera; CULQU, Culex quinquefasciatus; HYDVU, Hydra vulgaris; NEMVE, Nematostella vectensis; TRIAD Trichoplax adhaerens. b, Multiple sequence logo of the alignment performed in a generated using WebLogo 3 (http://weblogo.threeplusone.com)⁵³. c, HCT-116 cells were infected with lentiviruses expressing either FFSS-tagged empty vector (EV) or FFSS-tagged wild-type or mutant cyclin D1. Cells were treated with MLN4924 before lysis and affinity purification (AP) with an anti-STREP resin. Proteins were immunoblotted as indicated. d, HEK 293T cells were transiently transfected with plasmids expressing either SF-tagged empty vector or SF-tagged D-type cyclins wild-type or mutants. Cells were treated with MLN4924 before lysis and immunoprecipitation with an anti-Flag resin. Proteins were immunoblotted as indicated. e, HCT-116 cells were infected with retroviruses expressing FFSS-tagged wild-type or mutant cyclin D1. Cells were treated with cycloheximide for the indicated times before collection. Cell extracts were immunoblotted as indicated. f, HCT-116 cells were infected with retroviruses expressing FFSS-tagged wild-type or mutant cyclin D2. Cells were treated with cycloheximide for the indicated times before collection. Cell extracts were immunoblotted as indicated. g, HCT-116 cells were infected with retroviruses expressing FFSS-tagged wild-type or mutant cyclin D3. Cells were treated with cycloheximide for the indicated times before collection. Cell extracts were immunoblotted as indicated. h, HCT-116 cells were transfected with vectors expressing SF-tagged CDC25A, SF-tagged p27, SF-tagged cyclin D1, SF-tagged cyclin D1(T286A) or empty vector. Cells were treated with MLN4924 for 4 h before collection. Lysates were used for an immunoprecipitation with an anti-Flag resin. Proteins were immunoblotted as indicated. i, HCT-116 cells were transfected with vectors expressing HA-tagged FBXO4, SF-tagged TRF1, SF-tagged cyclin D1, SF-tagged cyclin D1(T286A) or empty vector. Cells were treated with MLN4924 for 4 h before collection. Lysates were used for an affinity purification with an anti-Strep resin. Proteins were immunoblotted as indicated. The asterisk indicates a non-specific band. j, HCT-116 cells were transfected with vectors expressing HA-tagged FBXW8, SF-tagged IRS1, SF-tagged cyclin D1, SF-tagged cyclin D1(T286A) or empty vector. Cells were treated with MLN4924 for 4 h before collection. Lysates were used for an immunoprecipitation with an anti-Flag resin. Proteins were immunoblotted as indicated. k, Parental and 2×Flag–mAID–AMBRA1 HCT-116 cells were lysed and their extracts were used for an immunoprecipitation with an anti-Flag resin. Proteins were immunoblotted as indicated. l, One clone of 2×Flag–mAID–AMBRA1 HCT-116 cells (clone F1) was infected with retroviruses expressing GFP-CCND1 or GFP-CCND1(T286A). Cells were treated with doxycycline for 12 h, before exposure to auxin for 4 h before collection as indicated. Control cells were treated with DMSO. Cell extracts were immunoblotted as indicated. m, Parental and AMBRA1^−/− HCT-116 and U-2 OS cells were lysed and their extracts were blotted as indicated. The accumulation of cyclin D1 and phosphorylated cyclin D1 (T286) is reported as their corresponding intensity ratios between AMBRA1^−/− and parental cells. Data are mean ± s.e.m. (n = 3 biological replicates per group). n, Three different AMBRA1^−/− HCT-116 clones (clones D2, F11 and G7), 2×Flag-mAID-AMBRA1 HCT-116 cells (clone F1) and HCT-116 cells transfected with a non-targeting siRNA or an siRNA against AMBRA1 were lysed and their extracts were immunoblotted as indicated (either by employing fluorescently labelled secondary antibodies or by enhanced chemiluminescence). Untransfected CCND1^−/− HCT-116 cells or CCND1^−/− HCT-116 cells transfected with vectors expressing untagged cyclin D1(WT) or untagged cyclin D1(T286A) were used as control. Where indicated (black triangles), samples were serially diluted before electrophoresis and immunoblotting for easier visual comparison of cyclin D1 and phosphorylated cyclin D1 (T286) levels between groups. The analysis of normalized phosphorylated cyclin D1 (T286) over total cyclin D1 fluorescent intensity levels are provided as bar graphs. The ratios were calculated from lane 2 and lane 4 from each biological group in the fluorescent immunoblot. o, HCT-116 cells were infected with lentiviruses expressing FFSS-tagged cyclin D1 or FFSS-tagged cyclin D1(T286A). Cells were treated with DMSO (N/T) or MLN4924 for 4 h before lysis and immunoprecipitation with an anti-Flag resin, followed by elution with a 3×Flag peptide. Eluted samples were then analysed by LC-MS/MS for the identification of post-translational modifications. The table reports all identified sites in wild-type cyclin D1 and cyclin D1(T286A) that were modified with a di-Gly/114.0429 signature. p, Synthetic, unphosphorylated (amino acids PKAAEEEEEEEEEVDLACTPTDVRDVDI) and phosphorylated (T286) (amino acids PKAAEEEEEEEEEVDLAC-pT-PTDVRDVDI) peptides (2.5 μg each) corresponding to the extreme C terminus of cyclin D1 were spotted on a nitrocellulose membrane before immunoblotting with an anti-phosphorylated cyclin D1 (T286) antibody. q, Synthetic, phosphorylated (T286) peptide corresponding to the sequence of cyclin D1 indicated in p was incubated with in vitro transcribed and translated AMBRA1 from rabbit reticulocytes extracts for the indicated times in the presence of wild-type ubiquitin (Ub) or a mutant of ubiquitin [Ub(K0)] in which all lysine residues have been mutated to arginine. Reactions were stopped with Laemmli buffer. Proteins were immunoblotted as indicated. Unless otherwise noted, experiments were performed at least three independent times.