Abstract
Disabling cellular defense mechanisms is essential for induction of apoptosis. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. Here, we show that genetic deletion of TRB3 results in basal activation of AKT, preserves mitochondrial integrity, and confers resistance against cytokine-induced pancreatic beta cell death. Mechanistically, we find that TRB3 stabilizes MLK3, most likely by suppressing AKT-directed phosphorylation, ubiquitination, and proteasomal degradation of MLK3. Accordingly, TRB3(-/-) islets show a decrease in both the amplitude and duration of cytokine-stimulated MLK3 induction and JNK activation. It is well known that JNK signaling is facilitated by a feed forward loop of sequential kinase phosphorylation and is reinforced by a mutual stabilization of the module components. The failure of TRB3(-/-) islets to mount an optimal JNK activation response, coupled with the ability of TRB3 to engage and maintain steady state levels of MLK3, recasts TRB3 as an integral functional component of the JNK module in pancreatic beta cells.
Highlights
MLK3 induces TRB3 to inhibit AKT and compromise beta cell survival
TRB3 Is Required for Optimal MLK3-JNK Activation—We have previously reported that the MAP3K MLK3 and the pseudokinase TRB3 are both induced by IL-1 and function in concert to diminish survival mechanisms in cytokine-treated beta cells
To examine whether TRB3 directly contributes to the dampening effect of AKT on MLK3 levels, we transfected Min6 cells with equal amounts of GST-MLK3 in the presence of kinase dead or constitutively active myristoylated AKT
Summary
Results: AKT regulates novel phosphorylation, ubiquitination, and degradation of MLK3, explaining why TRB3Ϫ/Ϫ islets display attenuated MLK3/JNK activation and beta cell death. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. In the beta cell, MLK3 engagement stabilizes TRB3 protein levels to suppress AKT survival kinase and facilitate downstream proinflammatory effects of IL-1 [6]. Our results show that the ubiquitin acceptor site instrumental for triggering degradation of MLK3, coincides with the acceptor site for TRAF6-dependent Lys-63linked ubiquitination and activation of MLK3 This site-specific convergence of functionally divergent ubiquitination events, shows that ubiquitination of MLK3 (Lys-48 versus Lys-63) plays a crucial role in driving cytokine-mediated beta cell death. These data suggest that factors responsible for fine-tuning the dynamics of cytokine signaling can be exploited for therapeutic intervention
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