We have characterized the physiological NHE3 activation and translocation to the surface that follows initiation of Na+
-glucose cotransport as a process that depends on ezrin phosphorylation (25
). This ezrin phosphorylation requires upstream p38 MAP kinase activation but is unlikely to be mediated directly by p38 MAP kinase, because ezrin threonine 567 lacks the proline residues that are typically present within p38 MAP kinase targets. Rho kinase, PKA, and some PKC isoforms have been reported to phosphorylate ezrin or other ERM-related proteins (9
). However, rho kinase (27
) or PKA inhibition does not prevent NHE3 activation after initiation of Na+
-glucose cotransport, and both PKA and PKC activation are well known to reduce NHE3 activity and surface expression (30
). Thus, we sought an alternative kinase that might be directly responsible for ezrin phosphorylation in this physiologically relevant model of NHE3 activation. We performed in silico
analysis, which suggested Akt might phosphorylate ezrin at threonine 567. Akt has not been previously reported as a regulator of ezrin phosphorylation. However, Akt has been associated with translocation of NHE3 to the surface by undefined mechanism(s) (26
). We therefore tested the hypothesis that the role of Akt might be to phosphorylate ezrin at threonine 567.
We found that Akt is activated after initiation of Na+
-glucose cotransport with kinetics that parallel those of ezrin phosphorylation. We therefore asked if p38 MAP kinase inhibition could prevent Akt activation, because we have previously shown that, in this model, ezrin phosphorylation requires upstream p38 MAP kinase activation. The data show that, like ezrin phosphorylation, Akt phosphorylation requires p38 MAP kinase activity. This is consistent with previous studies in smooth muscle and mesenchymal cells suggesting a relationship between p38 MAP kinase and downstream Akt activation (45
), including a recent report that p38 MAP kinase and its downstream effector MAPKAP-2 can mediate Akt activation (46
). A separate study, also in muscle cells, demonstrated that Akt serine 473 is a substrate for MAPKAP-2 phosphorylation (31
). Our data suggest that this signaling pathway linking p38 MAP kinase to Akt may also be active in epithelia.
To test whether Akt could directly phosphorylate ezrin, we performed in vitro
kinase assays using immunopurified active Akt and recombinant ezrin. These studies show that Akt can phosphorylate ezrin at threonine 567. This result was predicted by the in silico
analysis, but contrasts with previous data suggesting that, in regulation of cell survival, cells expressing dominant-negative ezrin fail to activate Akt (32
). Thus, we examined Akt activation in cells expressing dominant-negative ezrin. This dominant-negative Nter ezrin expression is sufficient to block NHE3 translocation and activation (25
). However, the data show that Akt is activated normally after initiation of Na+
-glucose cotransport in monolayers of cells expressing dominant-negative ezrin. Thus, we conclude that Akt activation after initiation of Na+
-glucose cotransport does not require ezrin activation.
Based on these data, we concluded that Akt is able to phosphorylate ezrin at threonine 567. However, we also wanted to know whether Akt is responsible for the ezrin phosphorylation that occurs in living cells after initiation of Na+-glucose cotransport. Thus, we used three complementary approaches to determine whether Akt is responsible for ezrin phosphorylation and downstream NHE3 translocation and activation. First, we inhibited the upstream Akt regulator PI3 kinase. This reduced Akt phosphorylation to undetectable levels and completely blocked increases in ezrin phosphorylation. Likewise, pharmacological inhibition of Akt blocked increases in ezrin phosphorylation. Finally, after showing that Akt2 is the predominant isoform in Caco-2 intestinal epithelial cells, we used siRNA to knock down Akt2 expression. This also prevented increases in ezrin phosphorylation after initiation of Na+-glucose cotransport. In addition, either pharmacological inhibition or siRNA knockdown of Akt prevented both NHE3-dependent cytoplasmic alkalinization and NHE3 surface translocation. Thus, Akt2 is required for ezrin phosphorylation as well as downstream NHE3 translocation and activation after initiation of Na+-glucose cotransport.
Although the role of ezrin in NHE3 regulation has generally been considered to be mediated through NHE3 regulatory factors (21
), cell surface NHE3 expression is not altered by expression of mutant NHERF-1 (47
), and brush border NHE3 content is not reduced in NHERF-1 knockout mice (49
). Thus, another mechanism of ezrin-dependent NHE3 regulation (e.g.
a direct interaction mediated by a putative ezrin-binding site at the C-terminal of NHE3 (21
)) may be involved. Although Akt has been previously associated with translocation of NHE3 (24
), the mechanisms of this regulation are incompletely defined. For example, EGF-mediated stimulation of PI3-kinase results in increased Akt activity within early endosome and brush border plasma membrane fractions that correlates with increased total and phosphorylated Akt2 at those sites. This PI3-kinase-dependent increase in Akt activity correlated with translocation of NHE3 to the brush border (50
). Separate studies have shown that a significant fraction of brush border NHE3 is found in detergent-insoluble lipid rafts (50
) that also contain Akt2 (42
) and ezrin (51
). Until now, however, a functional relationship between ezrin and Akt2 has not been proposed. The data presented here suggest that one critical role of Akt2 in NHE3 regulation is to phosphorylate ezrin. Although other Akt2 targets may still be involved in this process, it is notable that dominant-negative ezrin prevents NHE3 translocation without altering Akt2 activation. Thus, when considered as a whole, these data and those from previous studies suggest that Akt2 works through ezrin to effect translocation of NHE3 from an intracellular pool to lipid raft domains within the brush border.
This Akt- and ezrin-dependent NHE3 translocation may also be relevant to studies of other transporters and receptors and explain otherwise unrelated data from disparate systems. For example, GLUT4 is translocated from intracellular pools to the adipocyte plasma membrane by Akt- and p38 MAP kinase-dependent mechanisms after insulin stimulation (53
). In these cells, PI3 kinase mediates Akt activation (56
), but PI3 kinase-independent events are also required (58
). These include activation of the small GTPase TC10, whose activity within lipid rafts regulates cortical actin (59
) and is essential for docking and fusion of GLUT4-containing vesicles with the plasma membrane (60
). Thus, aspects of signal transduction pathways that mediate both NHE3 and GLUT4 trafficking and activation include p38 MAP kinase, PI3 kinase, Akt2, and actin. Although no role for ezrin in GLUT4 translocation has been reported, there are also no reports that it has been considered. Taken together, these data suggest the existence of a signalosome including p38 MAP kinase, PI3-kinase, Akt2, and ezrin that may regulate traffic between the plasma membrane and intracellular vesicles.
In summary, we show here that Akt2 phosphorylates ezrin after initiation of Na+-glucose cotransport. Akt2 activation requires p38 MAP kinase activity but is independent of ezrin function. Akt2-dependent ezrin phosphorylation is required for subsequent NHE3 translocation and activation. Thus, these studies identify ezrin activation as a critical role of Akt2 in NHE3 regulation. In addition to showing that Akt phosphorylates ezrin in this pathway, these data may also lead to understanding of other examples of rho-kinase independent ezrin activation as well as Akt-dependent trafficking events.