Plasma Membrane Subdomain Compartmentalization Contributes to Distinct Mechanisms of Ceramide Action on Insulin Signaling

International audienc

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein–protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity

Ceramides contained in LDL are elevated in type 2 diabetes and promote inflammation and skeletal muscle insulin resistance

Dysregulated lipid metabolism and inflammation are linked to the development of insulin resistance in obesity, and the intracellular accumulation of the sphingolipid ceramide has been implicated in these processes. Here, we explored the role of circulating ceramide on the pathogenesis of insulin resistance. Ceramide transported in LDL is elevated in the plasma of obese patients with type 2 diabetes and correlated with insulin resistance but not with the degree of obesity. Treating cultured myotubes with LDL containing ceramide promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation compared with LDL deficient in ceramide. LDL-ceramide induced a proinflammatory response in cultured macrophages via toll-like receptor-dependent and -independent mechanisms. Finally, infusing LDL-ceramide into lean mice reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle. These newly identified roles of LDL-ceramide suggest that strategies aimed at reducing hepatic ceramide production or reducing ceramide packaging into lipoproteins may improve skeletal muscle insulin action

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the “paradoxical” Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus