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.     

Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells

Angiotensin II activates a variety of signaling pathways in vascular smooth muscle cells (VSMCs), including the MAPKs and Akt, both of which are required for hypertrophy. However, little is known about the relationship between these kinases or about the upstream activators of Akt. In this study, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive kinase p38 MAPK and its substrate MAPKAPK-2 mediate Akt activation in VSMCs. In unstimulated VSMCs, Akt and p38 MAPK are constitutively associated and remain so after angiotensin II stimulation. Inhibition of p38 MAPK activity with SB-203580 dose-dependently inhibits Akt phosphorylation on Ser⁴⁷³, but not Thr³⁰⁸. Angiotensin II-induced phosphorylation of MAPKAPK-2 is also attenuated by SB-203580, as well as by inhibitors of ROS. In addition, angiotensin II stimulates the association of MAPKAPK-2 with the Akt-p38 MAPK complex, and an in vitro kinase assay shows that MAPKAPK-2 immunoprecipitates of VSMC lysates phosphorylate recombinant Akt in an angiotensin II-inducible manner. Finally, intracellular delivery of a MAPKAPK-2 peptide inhibitor blocks Akt phosphorylation on Ser⁴⁷³. These results suggest that the p38 MAPK-MAPKAPK-2 pathway mediates Akt activation by angiotensin II in these cells by recruiting active MAPKAPK-2 to a signaling complex that includes both Akt and p38 MAPK. Through this mechanism, p38 MAPK confers ROS sensitivity to Akt and facilitates downstream signaling. These results provide evidence for a novel signaling complex that may help to spatially organize hypertrophy-related, ROS-sensitive signaling in VSMCs. mitogen-activated protein kinase; reactive oxygen species     

The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival

Apoptosis results in cell shrinkage and intracellular acidification, processes opposed by the ubiquitously expressed NHE1 Na(+)/H(+) exchanger. In addition to mediating Na(+)/H(+) transport, NHE1 interacts with ezrin/radixin/moesin (ERM), which tethers NHE1 to cortical actin cytoskeleton to regulate cell shape, adhesion, motility, and resistance to apoptosis. We hypothesize that apoptotic stress activates NHE1-dependent Na(+)/H(+) exchange, and NHE1-ERM interaction is required for cell survival signaling. Apoptotic stimuli induced NHE1-regulated Na(+)/H(+) transport, as demonstrated by ethyl-N-isopropyl-amiloride-inhibitable, intracellular alkalinization. Ectopic NHE1, but not NHE3, expression rescued NHE1-null cells from apoptosis induced by staurosporine or N-ethylmaleimide-stimulated KCl efflux. When cells were subjected to apoptotic stress, NHE1 and phosphorylated ERM physically associated within the cytoskeleton-enriched fraction, resulting in activation of the pro-survival kinase, Akt. NHE1-associated Akt activity and cell survival were inhibited in cells expressing ERM binding-deficient NHE1, dominant negative ezrin constructs, or ezrin mutants with defective binding to phosphoinositide 3-kinase, an upstream regulator of Akt. We conclude that NHE1 promotes cell survival by dual mechanisms: by defending cell volume and pH(i) through Na(+)/H(+) exchange and by functioning as a scaffold for recruitment of a signalplex that includes ERM, phosphoinositide 3-kinase, and Akt.     

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.     

Osmotic cell shrinkage activates ezrin/radixin/moesin (ERM) proteins: activation mechanisms and physiological implications

Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr(567/564/558) (ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P(2) depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na(+)/H(+) exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P(2)-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.     

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin,ERK, and p38 MAPK

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.     

The Na+/H+ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation

Na⁺/H⁺ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

beta-Amyloid peptide induces formation of actin stress fibers through p38 mitogen-activated protein kinase

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.     

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

Modulation of protein phosphatase 2a by adenosine A1 receptors in cardiomyocytes: role for p38 MAPK

Adenosine A1 receptor activation causes protein phosphatase 2a (PP2a) activation in ventricular myocytes. This attenuates beta-adrenergic functional effects in the heart (Liu Q and Hofmann PA. Am J Physiol Heart Circ Physiol 283: H1314-H1321, 2002). The purpose of the present study was to identify the signaling pathway involved in the translocation/activation of PP2a by adenosine A1 receptors in ventricular myocytes. We found that N6-cyclopentyladenosine (CPA; an adenosine A1 receptor agonist)-induced PP2a translocation was blocked by p38 MAPK inhibition but not by JNK inhibition. CPA increased phosphorylation of p38 MAPK, and this effect was abolished by pertussis toxin and inhibitors of the cGMP pathway. Moreover, CPA-induced PP2a translocation was blocked by inhibition of the cGMP pathway. Guanylyl cyclase activation mimicked the effects of CPA and caused p38 MAPK phosphorylation and PP2a translocation. Finally, CPA-induced dephosphorylations of troponin I and phospholamban were blocked by pertussis toxin and attenuated by p38 MAPK inhibition. These results suggest that adenosine A1 receptor-mediated PP2a activation uses a pertussis toxin-sensitive Gi protein-guanylyl cyclase-p38 MAPK pathway. This proposed, novel pathway may play a role in acute modulation of cardiac function.     

NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3

The sodium-hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na(+)/H(+) exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1-355), the C-terminal domain, NHERF(147-355), and NHERF(1-325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1-146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1-355) but not with NHERF(1-325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1-355) or NHERF(147-355) but had no effect on the formation of NHE3-NHERF or NHERF-ezrin complexes. Na(+)/H(+) exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1-146) or NHERF(1-325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na(+)/H(+) exchange.     

Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1) during osmotic cell shrinkage

The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Regulation of the human Na⁺-dependent glucose cotransporter hSGLT2

The human Na(+)-glucose cotransporter SGLT2 is expressed mainly in the kidney proximal convoluted tubule where it is considered to be responsible for the bulk of glucose reabsorption. Phosphorylation profiling has revealed that SGLT2 exists in a phosphorylated state in the rat renal proximal tubule cortex, so we decided to investigate the regulation of human SGLT2 (hSGLT2) by protein kinases. hSGLT2 was expressed in human embryonic kidney (HEK) 293T cells, and the activity of the protein was measured using radiotracer and whole cell patch-clamp electrophysiology assays before and after activation of protein kinases. 8-Bromo-adenosine cAMP (8-Br-cAMP) was used to activate protein kinase A, and sn-1,2-dioctanoylglycerol (DOG) was used to activate protein kinase C (PKC). 8-Br-cAMP stimulated D-[α-methyl-(14)C]glucopyranoside ([(14)C]α-MDG) uptake and Na(+)-glucose currents by 200% and DOG increased [(14)C]α-MDG uptake and Na(+)-glucose currents by 50%. In both cases the increase in SGLT2 activity was marked by an increase in the maximum rate of transport with no change in glucose affinity. These effects were completely negated by mutation of serine 624 to alanine. Insulin induced a 250% increase in Na(+)-glucose transport by wild-type but not S624A SGLT2. Parallel studies confirmed that the activity of hSGLT1 was regulated by PKA and PKC due to changes in the number of transporters in the cell membrane. hSGLT1 was relatively insensitive to insulin. We conclude that hSGLT1 and hSGLT2 are regulated by different mechanisms and suggest that insulin is an SGLT2 agonist in vivo.