Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

Abstract

Initiation of Na؉ -glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na ؉ -glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na ؉ -glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na ؉ -glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation.

Sodium-Dependent Glucose Transporter-1 as a Novel Immunological Player in the Intestinal Mucosa

“…6C 1 ). When HT29 cells were incubated with LY-294002, previously used as a pharmacological inhibitor of Akt phosphorylation (22), plus glucose and LPS, we observed a nuclear translocation of NF-B comparable to LPS-treated cells, confirming the role of Akt phosphorylation in the pathway leading to NF-B inhibition by glucose (Fig. 6C 2 ).…”

“…Akt phosphorylation may be induced by SGLT-1 activation; in fact, Na ϩ -glucose cotransport mediated by SGLT-1 leads to a complex signal transduction pathway in which activation of p38 MAPK leads to Akt activation. Hu et al (23) have reported that the transport by the apical Na ϩ -glucose cotransporter SGLT-1 triggers translocation of NHE3 to the plasma membrane by activation of p38 MAPK, Akt2, and ezrin, whereas Shiue et al (22) suggested that inhibition of Akt using inhibitors prevents ezrin phosphorylation after initiation of Na ϩ -glucose cotransport. Moreover, Guha and Mackman (25) recently reported that the PI3K pathway in monocytes suppresses both MAPKs and NF-B cascades in response to LPS, resulting in decreased production of TNF-␣.…”

“…Some authors have reported that SGLT-1 activation may induce Akt phosphorylation, coordinating the functional absorption of enterocytes by luminal nutrients (22)(23)(24). It is instead well known that Akt may negatively regulate TLR signaling, inhibiting NF-B translocation (25).…”

Molecular Basis of Epithelial Barrier Regulation

“…34 These data, therefore, showed that MLCK-mediated MLC phosphorylation was necessary for Na ϩ -glucose cotransport-induced tight junction regulation. However, as subsequent work has identified a complex cascade of kinases that are activated by Na ϩ -glucose cotransport, [35][36][37][38] it was not clear whether MLCKmediated MLC phosphorylation alone was sufficient to regulate tight junction permeability.…”

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

“…None of the polarized intestinal epithelial cell lines available today express detectable levels of transepithelial Na + -dependent glucose transport, which has been attributed to a deficiency of surface expression of SGLT-1 ( [Turner et al, 1996] and [Kipp et al, 2003]). In these transfected cells, the expression of SGLT-1 protein on the apical membrane is associated with functional Na + -dependent uptake of glucose, and physiological transduction pathways in response to SGLT-1 activation ( [Turner et al, 1996], [Shiue et al, 2005] and [Hu et al, 2006]). These cells were seeded in 8-well chamber slides (4 × 10 5 cells/well; Lab-Tek, Nalge Nunc, Rochester, NY), 24-well plates (10 6 cells/well; Costar, Corning Inc., Corning, NY), 6-well plates (6 × 10 6 cells/well, Costar) and 12-well transwells, which contained 1 cm 2 semipermeable filter membrane with 0.4 μm pores (10 6 cells/well, Costar) and grown to confluency for 1 week at 37 °C with 5% CO 2 and 96% humidity for experiments.…”