Erythrocyte Spectrin Is an E2 Ubiquitin Conjugating Enzyme

Abstract

The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.

Ubiquitination of red blood cell α‐spectrin does not affect heterodimer formation

“…Interestingly, the aV domain contains spectrin repeats 20-22 that our laboratory indicated as containing likely E2/E3 and ubiquitin acceptor sites [17]. Mature RBCs have lost their nucleus and other organelles including most proteosomes, and, therefore, ubiquitination of spectrin in the erythrocyte probably primarily serves regulatory roles in protein-protein interactions.…”

“…Galluzi et al [9,10] have demonstrated two ubiquitination sites within a-spectrin: one in a-spectrin repeat 17 and the other in a-spectrin repeats 20 and 21. It has been further demonstrated by our laboratory that a 0 -spectrin represents ubiquitin attached to a-spectrin via a thioester bond, and this ubiquitin spectrin adduct has E2 ubiquitin conjugating activity and can transfer the ubiquitin (possibly via an E3 site) to an intramolecular DTT insensitive target site in a-spectrin repeats 20/21 [17].…”

“…Human erythrocyte ghosts and spectrin were isolated as described [17]. The aand b-spectrin monomers were isolated as previously described by our laboratory [18].…”

“…An anti-rabbit IgG was used as the secondary antibody (1/8,000). The a-and b-spectrin antibodies were prepared as described [17]. A peptide-specific antibody against residues 16-34 of ubiquitin was prepared commercially (Biosynthesis Inc., Lewisville, TX).…”

“…This modified a-spectrin migrates more slowly than unmodified a-spectrin on SDS-PAGE in the absence of DTT and is observed in lower amounts in sickle cell RBCs [17]. Galluzi et al [9,10] have demonstrated two ubiquitination sites within a-spectrin: one in a-spectrin repeat 17 and the other in a-spectrin repeats 20 and 21.…”