The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured ␤ cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.The homeodomain protein PDX1 has been shown to be a critical regulator of pancreatic development, in both humans and in mice (1, 2). Whereas genetic ablation or the total functional inhibition of PDX1 results in pancreatic agenesis, haploinsufficiency of PDX1 in humans leads to diabetes (3) and is associated with diminished glycemic control in mice (4, 5). Loss of a single allele of Pdx1 results in increased beta cell death (6) and a diminished capacity to mount a compensatory response in some models of insulin resistance (7). Thus, the beta cell is highly sensitive to total cellular levels of PDX1 protein.The mechanisms by which PDX1 may regulate glucose homeostasis have been widely examined. In addition to regulating the insulin gene (8, 9), PDX1 has been shown to activate Glut2 and glucokinase genes, two key players required for glucose sensing (10, 11). PDX1 can also impact glucose sensing by influencing expression of mitochondrial metabolic pathways (12, 13). Accordingly, proteins important in glucose sensing are down-regulated in Pdx1 ϩ/Ϫ mice (5, 7). In the beta cell, PDX1 is thought to be a direct mediator of glucose. Several studies have suggested that glucose-stimulated phosphorylation of PDX1 impacts DNA binding (14, 15) and the nucleocytoplasmic shuttling of PDX1 (16). Glucose-dependent phosphorylation has also been suggested to increase the transactivation potential of PDX1 by increasing recruitment of chromatin-modifying proteins such as p300 (17,18) or by decreasing binding with histone deacetylases (19). Other studies suggest direct, glucose-dependent phosphorylation of PDX1 by specific kinases, including ERK 2 1 and 2 (20), and perhaps p38 MAPK (14, 16). However, to date, the specific amino acid residues at which glucose-induces PDX1 phosphorylation, within the context of the cell, are not known.The N terminus of PDX1 harbors a strong transactivation domain, flanked by the DNA-binding homeodomain (8,21,22). The C terminus of PDX1 ...

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of nonobese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.In type 1 diabetes, the autoimmune destruction of pancreatic beta cells is driven by leukocyte infiltration and the damaging effects of locally secreted cytokines. Cytokines activate MAPKs 3 JNK and p38, via signaling modules that involve the sequential activation of a MAP3K, MAP2K, and MAPK, all scaffolded by a single protein (1). The existence of several families of MAP3Ks raises the possibility that each MAP3K may be activated by specific classes of stimuli. The serine-threonine MAP3K mixed lineage kinase-3 (MLK3) is activated by cytokines (2, 3) and assembles a signaling module consisting of MKK7, JNK, and the scaffold protein JIP1 (4, 5). Fibroblasts with a targeted deletion of either MKK7 or MLK3 are attenuated in their response to cytokines (6, 7). Elevation of MLK3 has been linked to induction of apoptosis in neurons (8 -10), and inhibition of MLKs can delay progression of neurodegenerative diseases (reviewed in Ref. 11 and studies quoted therein). The striking parallels between the beta cell and neuronal phenotypes, coupled with the ability of cytokines to activate MLK3, prompted us to examine whether MLKs participate in cytokine-induced beta cell death.Here we show that MLK3 is markedly elevated in leukocyteinfiltrated islets of the non-obese type 1 diabetic (NOD) mouse. To investigate the potential role of MLK3 in beta cell death, we devised an ex vivo system for co-culture of primary islets with immune-activated splenocytes. Compared with static culture with purified cytokines, this system is likely to be more representative of the milieu encountered by islets in autoimmune diabetes. We observed rapid activation of MLK3, and MLK3 was required for cytokine-mediated apoptosis via BAX, a proapoptotic member of the BCL-2 protein family. MLK3 mediated its effects via the pseudokinase TRB3 (TRIBBLES homolog 3), originally identified as an inducible factor in neuronal cell death (12) and subsequently shown to be a potent negative regulator of the prosurvival kinase Akt (13). We fo...