Akt2 phosphorylates ezrin to trigger NHE3 translocation and 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.
Shiue
Harn
H
Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA.
Musch
Mark W
MW
Wang
Yingmin
Y
Chang
Eugene B
EB
Turner
Jerrold R
JR
D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D019295:Computational Biology; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D005947:Glucose; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010750:Phosphoproteins; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D012964:Sodium; D051269:Sodium-Glucose Transporter 1; D017923:Sodium-Hydrogen Exchangers; D048051:p38 Mitogen-Activated Protein Kinases; D000099149:Ezrin; C494918:AKT1 protein, human; C494921:AKT2 protein, human