Analysis of the role of ubiquitin-interacting motifs in ubiquitin binding and ubiquitylation.
The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIM-dependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.
Miller
Stephanie L H
SL
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Malotky
Erica
E
O'Bryan
John P
JP
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000067699:Ataxin-3; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004622:Embryo, Mammalian; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D010455:Peptides / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
C492511:ANKIB1 protein, human; D033942:Adaptor Proteins, Vesicular Transport; C121958:DNAJB2 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; C114154:epsin; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C085706:FLAG peptide; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; C482576:MAP3K1 protein, human; C092341:ATXN3 protein, human; D000067699:Ataxin-3