NDRG1-PTEN association modulates PTEN C-terminal tail phosphorylation in insulin-resistant neurons

N-myc Downstream Regulated Gene-1 (NDRG1), implicated in the regulation of the PI3K-Akt pathway, regulates the expression of Phosphatase and TENsin homolog (PTEN), a key insulin signaling regulator. However, NDRG1's role in the insulin signaling pathway is not understood. This study aimed to clarify whether and how NDRG1 regulates PTEN C-terminal phosphorylation at residues S380/T382/T383 in the context of neuronal insulin signaling and resistance. It was found that NDRG1 associates with PTEN and regulates its phosphorylation at residues S380/T382/T383 in mouse hippocampal neurons. Co-immunoprecipitation and immunofluorescence microscopy indicated that pairwise associations between NDRG1-PTEN and Protein Phosphatase 2A (PP2A)-PTEN are upregulated by insulin stimulation and are further augmented in insulin-resistance. Deletion of the NDRG1 C-terminal region markedly reduced detectable PTEN co-immunoprecipitation, suggesting a role for this region in NDRG1-PTEN association. Silencing NDRG1 increased PTEN C-terminal phosphorylation at residues S380/T382/T383. Additionally, data from PP2A-specific inhibitor and siRNA support the involvement of PP2A in PTEN C-terminal dephosphorylation. We also utilized a phospho-dead NDRG1 mutant to demonstrate that phosphorylation of NDRG1 at T346 plays a role in the modulation of PTEN C-terminal phosphorylation. Thus, our results suggest that NDRG1 modulates PTEN C-terminal phosphorylation at residues S380/T382/T383 in neuronal cells and neuronal insulin-resistance affects this regulation by NDRG1.