Lysine 269 is essential for cyclin D1 ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ligase and subsequent proteasome-dependent degradation.
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.
Barbash
O
O
Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA.
Egan
E
E
Pontano
L L
LL
Kosak
J
J
Diehl
J A
JA
D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000502:physiology*; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D018411:Spodoptera; D054875:Ubiquitination / Q000502:physiology; D038203:alpha-Crystallin B Chain / Q000378:metabolism
C530105:Ccnd1 protein, mouse; D044783:F-Box Proteins; C515213:Fbx4 protein, mouse; D050505:Mutant Proteins; D038203:alpha-Crystallin B Chain; D019938:Cyclin D1; D001120:Arginine; D044843:SKP Cullin F-Box Protein Ligases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine