Lysine 63-linked Ubiquitination Modulates Mixed Lineage Kinase-3 Interaction with JIP1 Scaffold Protein in Cytokine-induced Pancreatic β Cell Death

Background: Cytokines activate MLK3 to compromise mitochondrial integrity and promote pancreatic cell death. Results: Results suggest a model in which IL-1/TRAF6-mediated Lys-63-linked ubiquitination of MLK3 dissociates monomeric MLK3 from JIP1 for subsequent dimerization and activation. Conclusion: IL-1-stimulated Lys-63-linked ubiquitination activates MLK3, most likely by altering the dynamics of MLK-JNK-JIP module. Significance: These findings can be exploited for therapeutic intervention in diabetes. The mixed lineage kinase MLK3 plays a crucial role in compromising mitochondrial integrity and functions as a proapoptotic competence factor in the early stages of cytokine-induced pancreatic cell death. In an effort to identify mechanisms that regulate MLK3 activity in cells, we discovered that IL-1 stimulates Lys-63-linked ubiquitination of MLK3 via a conserved, TRAF6-binding peptapeptide motif in the catalytic domain of the kinase. TRAF6-mediated ubiquitination was required for dissociation of inactive monomeric MLK3 from the scaffold protein IB1/JIP1, facilitating the subsequent dimerization, autophosphorylation, and catalytic activation of MLK3. Inability to ubiquitinate MLK3, or the presence of A20, an upstream Lys-63-linked deubiquitinase, strongly curtailed the ability of MLK3 to affect the proapoptotic translocation of BAX in cytokine-stimulated pancreatic cells, an early step in the progression toward cell death. These studies suggest a novel mechanism for MLK3 activation and provide new clues for therapeutic intervention in promoting cell survival.