Ankyrin is a target of spectrin's E2/E3 ubiquitin-conjugating/ligating activity.
Ubiquitin is a small protein of 8.6 kDa molecular weight. When polyubiquitin is attached to target proteins, they are tagged for destruction by cytoplasmic organelles called proteasomes. We now know that ubiquitination of target proteins also regulates functions as diverse as the sorting of proteins to different intracellular destinations, cell signaling, cell division, gene transcription, and protein-protein interactions. The ubiquitination of target proteins requires a cascade of enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligating enzyme. Recently we have demonstrated that the red blood cell (RBC) membrane skeletal protein, spectrin, has E2/E3 enzymatic activities in its alpha-subunit, that can transfer ubiquitin to itself. We have now created a cell free assay using biotinylated ubiquitin that allows detection of target proteins by streptavidin peroxidase. This approach coupled with immunoprecipitation, purification and micro liquid chromatography coupled to tandem mass spectrometry has identified ankyrin as a target of spectrin's E2/E3 activity. Western blotting, with ubiquitin antibody, of purified ankyrin and its well characterized functional domains, has demonstrated that both the spectrin and band 3 binding domains are ubiquitinated in vivo.
Chang
T L
TL
Department of Molecular and Cell Biology, UTD Sickle Cell Disease Research Center, Institute of Biomedical Sciences and Technology, The University of Texas at Dallas, Richardson, TX 75083, USA.
Cubillos
F F
FF
Kakhniashvili
D G
DG
Goodman
S R
SR
D017487:Ankyrins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D011233:Precipitin Tests; D011485:Protein Binding; D013049:Spectrin / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D017487:Ankyrins; D014452:Ubiquitins; D013049:Spectrin; D044763:Ubiquitin-Conjugating Enzymes; D014357:Trypsin