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J. Biol. Chem., Vol. 280, Issue 2, 1688-1695, January 14, 2005

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Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation^*

Harn Shiue, Mark W. Musch¶, Yingmin Wang¶, Eugene B. Chang, and Jerrold R. Turner||

From the Departments of Pathology and Medicine, The University of Chicago, Chicago, Illinois 60637

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.

Received for publication, August 18, 2004 , and in revised form, November 3, 2004.

^* This work was supported by the National Institutes of Health (Grants R01-DK61931 to J. R. T. and R01-DK038510 to E. B. C.), The Crohn's Colitis Foundation of America (to J. R. T.), The University of Chicago Digestive Disease Center (P30-DK42086), and the University of Chicago Cancer Center (P30-CA14599). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both authors contributed equally to this work.

^|| To whom correspondence should be addressed: 5841 S. Maryland Ave., MC 1089, Chicago, IL 60637. Tel.: 773-702-2433; Fax: 773-834-5251; E-mail: jturner{at}bsd.uchicago.edu.

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