Background/Purpose:  ABCG2 is a high capacity urate secretory transporter of the renal proximal tubule. The common Q141K ABCG2 mutation causes gout in humans through an increased instability of the nucleotide-binding domain leading to enhanced degradation and reduced function.

Methods: Typical biochemical and cell biological techniques

Results:  Here, we found ABCG2 protein rescued from degradation with the proteasome inhibitor MG-132 is phosphorylated; raising the possibility that a phospho-degron regulates ABCG2 trafficking and expression. An in silicoanalysis of ABCG2 revealed a limited number of predicted phosphorylation sites, including S195, a serine conserved in the mammalian lineage. The upstream RXRXS represents a target motif for AKT1 and PKA, which both co-immunoprecipitated with ABCG2. Specifically, endogenous AKT1 pulled down both over expressed ABCG2 in HEK293 cells as well as endogenous ABCG2 in mouse kidney lysate. AKT1 and ABCG2 transcript co-localize in the proximal S2 segment of the mammalian nephron and inhibiting the AKT1 kinase cascade with PI3K inhibitor LY294002, or with growth factor receptor (RTK) inhibitor Vandetanib, dramatically up-regulated ABCG2 expression. Conversely, activating the AKT1 cascade with FBS down-regulated ABCG2 expression. Replacement of the S195 residue with a phosphomimetic aspartic acid resulted in significant reduction in ABCG2 expression, localization of ABCG2 to peri-nuclear compartments, and significant sensitivity to MG-132; confirming the S195 residue as a phospho-degron. Finally, a non-phosphorylatable S195A substitution led to the complete rescue of the Q141K gout mutant protein expression and trafficking.

Conclusion: Modeled ABCG2 structure indicates phosphorylation of the S195 residue may only be possible when the nucleotide-binding domains are separated, suggesting the S195 phospho-degron may be part of a novel regulatory mechanism for function and trafficking in ABC transporters. Funded by: American Heart Association 14SDG18060004 & Ardea BioSciences.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-of-phospho-degron-regulating-expression-of-urate-secretory-transporter-abcg2/