Initiation of Na ؉ -glucose cotransport in intestinal epithelial cells leads to activation of the apical Na ؉ -H ؉ exchanger NHE3 and subsequent increases in cytoplasmic pH (pH i). This process requires activation of p38 mitogen-activated protein (MAP) kinase, but additional signaling intermediates have not been identified. One candidate is the cytoskeletal linker protein ezrin, which interacts with NHE3 via specific regulatory proteins. The data show that initiation of Na ؉ -glucose cotransport resulted in rapid increases in both apical membrane-associated NHE3 and cytoskeletal-associated ezrin and occurred in parallel with ezrin phosphorylation at threonine 567. Phosphorylation at this site is known to activate ezrin and increase its association with actin. Consistent with a central role for ezrin activation in this NHE3 regulation, an Nterminal dominant negative ezrin construct inhibited both NHE3 recruitment and pH i increases after Na ؉ -glucose cotransport. Ezrin phosphorylation occurred in parallel with p38 MAP kinase activation, and the latter proceeded normally in cells expressing dominant negative ezrin. In contrast, inhibition of p38 MAP kinase prevented increases in ezrin phosphorylation after initiation of Na ؉ -glucose cotransport. Thus, ezrin phosphorylation after Na ؉ -glucose cotransport requires p38 MAP kinase activity, but p38 MAP kinase activation does not require ezrin function. These data describe a specific role for ezrin in the coordinate regulation of Na ؉ -glucose cotransport and Na ؉ -H ؉ exchange. Intact ezrin function is necessary for NHE3 recruitment to the apical membrane and NHE3-dependent pH i increases triggered by Na ؉ -glucose cotransport. The data also define a pathway of p38 MAP kinase-dependent ezrin activation.

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.