Akt2 phosphorylates ezrin to trigger NHE3 translocation and activation

J Biol Chem. 2005 Jan 14;280(2):1688-95. doi: 10.1074/jbc.M409471200. Epub 2004 Nov 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Caco-2 Cells
  • Computational Biology
  • Cytoplasm / metabolism
  • Cytoskeletal Proteins
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Glucose / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Membrane Glycoproteins / metabolism
  • Molecular Sequence Data
  • Monosaccharide Transport Proteins / metabolism
  • Mutation / genetics
  • Phosphoproteins / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphothreonine / metabolism
  • Protein Transport
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Sodium / metabolism
  • Sodium-Glucose Transporter 1
  • Sodium-Hydrogen Exchangers / metabolism*
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Cytoskeletal Proteins
  • Enzyme Inhibitors
  • Membrane Glycoproteins
  • Monosaccharide Transport Proteins
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • Sodium-Glucose Transporter 1
  • Sodium-Hydrogen Exchangers
  • ezrin
  • Phosphothreonine
  • Sodium
  • AKT1 protein, human
  • AKT2 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • p38 Mitogen-Activated Protein Kinases
  • Glucose