A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation.
The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.
Institut de Pharmacologie et de Biologie Structurale (IPBS) CNRS 205 Route de Narbonne and IPBS, Université Paul Sabatier Université de Toulouse, Toulouse France.
Clara F
CF
Burande
Mélina L
ML
Heuzé
Isabelle
I
Lamsoul
Bernard
B
Monsarrat
Sandrine
S
Uttenweiler-Joseph
Pierre G
PG
Lutz
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism; D064448:Filamins; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism; D016608:Talin / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
C437987:ASB2 protein, human; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D050826:Suppressor of Cytokine Signaling Proteins; C518832:TLN1 protein, human; D016608:Talin; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex