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Ts shown are representative of four independent experiments. Asterisks denote nonspecific
Ts shown are representative of 4 independent experiments. Asterisks denote nonspecific bands. B , relative band intensities, as determined by densitometric evaluation with the blot shown in a. Error bars represent the S.E. (n four).Expression of Crbn WT, but Not Crbn R422X, Rescues the Translational De-repression Induced by Crbn Deficiency–To further validate the functional role of Crbn in translational CaMK II Inhibitor list regulation by way of AMPK-mTOR signaling, we attempted to rescue the phenotype in the Crbn deficiency by exogenously expressing either Crbn WT or Crbn R422X (Fig. 8A). Constitutive activation of AMPK in Crbn / MEF cells was efficiently suppressed by exogenous expression of WT Crbn (Fig. 8B). The expression of Crbn WT was also accompanied by greater levels of P-S6, as determined by Western-blot evaluation (Fig. 8C), and larger levels of cap-dependent translation, as determined by the relative luciferase assay (Fig. 8D). The exogenous expression of R422X Crbn, nonetheless, did not suppress AMPK phosphorylation (Fig. 8B). Accordingly, S6 phosphorylation andtranslational de-repression weren’t observed upon expression of the mutant protein. These final results further demonstrate that constitutive activation of AMPK can be a direct and reversible cellular response induced solely by the loss of Crbn, and that the lack of the endogenous Crbn gene may be rescued by exogenous expression of Crbn WT, but not by Crbn truncated consequently of a nonsense mutation.DISCUSSION It can be extensively accepted that memory formation requires not only mRNA transcription but also production of new proteins (17, 18, 29, 30). As the central regulator of translational initiation, the mTOR cascade is required for synaptic plasticity and memory processes that happen to be dependent around the protein synthesisVOLUME 289 Quantity 34 AUGUST 22,23348 JOURNAL OF BIOLOGICAL CHEMISTRYDysregulation of AMPK-mTOR Signaling by a Mutant CRBNmachinery (15, 171). The activity of mTOR, in turn, could be modulated by quite a few upstream kinases, which includes AMPK. As the cellular energy sensor and a adverse regulator of anabolic processes, activated AMPK phosphorylates mTORC1 and suppresses the synthesis of new cellular proteins (34, 35). Here we show, for the first time, that the expression degree of CRBN, a unfavorable regulator of AMPK, can successfully modulate the mTOR pathway and cellular protein synthesis. We observed that deficiency of endogenous Crbn resulted in constitutive activation of AMPK, thereby suppressing overall protein synthesis (controlled by the mTOR pathway) within the mouse hippocampus (Figs. 2 and 4). Accordingly, ectopic expression of CRBN WT suppressed AMPK activity and activated the mTOR pathway in human neuroblastoma (Fig. 5). Furthermore, the AMPK-dependent suppression of protein translation in Crbn / MEF cells was rescued by exogenous expression of Crbn WT, resulting in inhibition of endogenous AMPK activity (Fig. 8). These findings not only strengthen the concept that CRBN is definitely an endogenous negative regulator of AMPK (four, five), but also present a testable hypothesis regarding the mechanism by which the nonsense mutation in CRBN causes mental deficit in humans (Fig. 9). Due to the fact its initial identification as a candidate protein involved in human mental deficit (1), the significance of CRBN in brain function was additional demonstrated making use of a mouse model in which forebrain-specific D4 Receptor Antagonist review deletion of Crbn resulted in significant learning and memory defects (16). Furthermore, in whole-body Crbn-deficient mice, we also observed serious de.