GSK3β Regulates Toxic Nucleophosmin (NPM)-T<sup>95</sup> Phosphorylation During Ischemic AKI
November 07, 2019 | 10:00 AM - 12:00 PM
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GSK3β Regulates Toxic Nucleophosmin (NPM)-T95 Phosphorylation During Ischemic AKI
AKI: Mechanisms - Primary Injury and Repair - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
- Wang, Zhiyong, Boston Medical Center, Boston, Massachusetts, United States
- Salih, Erdjan, Boston University Medical Center, Boston, Massachusetts, United States
- Bonegio, Ramon G., Boston Medical Center, Boston, Massachusetts, United States
- Havasi, Andrea, Boston University Medical Center, Boston, Massachusetts, United States
- Igwebuike, Chinaemere, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
- Schwartz, John H., Boston Medical Center-Evans Biomedical Center, Boston, Massachusetts, United States
- Borkan, Steven C., Boston Medical Center, Boston, Massachusetts, United States
Ramon G. Bonegio,
John H. Schwartz,
Steven C. Borkan,
GSK3β promotes regulated renal cell death partly by phosphorylating and activating Bax. Here, we document that GSK3β also phosphorylates and activates NPM, a key Bax chaperone, during ischemic renal injury in vitro and AKI in vivo. We hypothesize that: (1) antagonizing NPM phosphorylation is effective for ameliorating acute renal cell injury that contributes to organ failure during AKI and (2) phosphorylated NPM detects acute renal cell injury.
To determine the extent to which GSK3β mediates ischemia-induced NPM phosphorylation, constitutively active or inactive GSK3β mutant proteins were expressed in primary murine and human proximal tubule cells (PTEC). GSK3β was also subjected to pharmacologic inhibition by TDZD-8 or CRISPRi-mediated knockdown. GSK3β activity was estimated from steady state p-NPM-T95 content and correlated with intracellular NPM localization and cell survival.
Transfection of primary PTEC with constitutively active, inactive or wild type GSK3β resulted in an expected increase in total GSK3β content compared with empty vector. In contrast, CRISPRi caused an 80% reduction in GSK3β expression. TDZD-8 decreased GSK3β kinase activity without affecting its content. Only active GSK3β promoted NPM T95 phosphorylation, NPM translocation from the nucleus to the cytosol and positively correlated with cell death during ischemia. Both TDZD-8 and CRISPRi–mediated GSK3β knockdown significantly reduced NPM T95 phosphorylation and cell death induced by constitutively active GSK3β. Furthermore, T95 NPM is detectable in both urine and cortical kidney homogenates harvested from humans and mice within hours after acute renal ischemia.
GSK3β promotes ischemia-induced renal cell death by phosphorylating an activating NPM, an essential partner for Bax during regulated cell death. Thus, manipulation of NPM phosphorylation is likely to be an effective therapeutic maneuver for ameliorating AKI and phosphorylated NPM is a novel marker of acute renal cell injury.
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