AKT and GSK-3 Are Necessary for Direct Ezrin Binding to NHE3 as Part of a C-terminal Stimulatory Complex

Singh, Varsha; Lin, Rong; Yang, Jianbo; Cha, Boyoung; Sarker, Rafiquel; Tse, Chung Ming; Donowitz, Mark

doi:10.1074/jbc.m113.521336

Cited by 19 publications

(25 citation statements)

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“…However, since only a 13% reduction of CHP binding was demonstrated, cause and effect has not been established. In addition, this stimulatory signaling complex involves direct binding of ezrin to NHE3 amino acids 517, 521, and 528, which requires phosphorylation of amino acids 516 and 527 (human NHE3) by Akt and GSK-3, respectively, as well as CHP binding (38). This ezrin binding to NHE3 is required not only for basal NHE3 activity but for multiple examples of stimulated NHE3 activity, and it is possible that ezrin binding is disrupted by this polymorphism to explain the failure of dexamethasone to stimulate NHE3.…”

Section: C762mentioning

confidence: 99%

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Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

Zhu

Sarker

Horton

et al. 2015

American Journal of Physiology-Cell Physiology

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Zhu XC, Sarker R, Horton JR, Chakraborty M, Chen TE, Tse CM, Cha B, Donowitz M. Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity. Am J Physiol Cell Physiol 308: C758 -C766, 2015. First published February 25, 2015 doi:10.1152/ajpcell.00421.2014.-Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na ϩ /H ϩ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na ϩ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function.

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Section: C762mentioning

confidence: 99%

“…This regulation involves both increased and reduced NHE3 activity, as occurs sequentially after meals. Mutations in this region lead to dysregulation of NHE3 to physiological activators or inhibitors (27,38,44).…”

mentioning

confidence: 99%

Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

Zhu

Sarker

Horton

et al. 2015

American Journal of Physiology-Cell Physiology

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“…This motif is present in the Pro-rich sequence of L-type GATA-6 ( 32 PSTP and 35 PPSP). Phosphorylation of the C-terminal Ser-rich sequence motif of the Na + /H + exchanger (NHE3) by Akt and GSK-3 stimulates NHE3 activity through Ezrin binding to this motif [26]. The consensus sequence for the GSK-3 substrate (S/T)XXX(S/T) is also present in the Pro-rich segment and the Ser-cluster of L-type GATA-6 ( 33 STPPS 37 and 40 SSSSSS 45 , respectively).…”

Section: Discussionmentioning

confidence: 99%

Increased Electrophoretic Mobility of Long-Type GATA-6 Transcription Factor upon Substitution of Its PEST Sequence

Obayashi¹,

Takada²,

Ohashi³

et al. 2014

ABB

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The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu 31 -Cys 46 ), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant; the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA * Corresponding author. K. Obayashi et al. 1033responsive promoter.

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“…This regulatory effect requires amino acids at its C-terminal to interact with the CaMKII binding domain downstream of NHE3 aa 690 (Ser693, Ser694, and Ser810), which is part of the putative CaMKII phosphorylation consensus sequence [45,46]. Recently, it was demonstrated that 1) NHE3 basal activity is regulated by a signaling complex that is controlled by the sequential effects of two kinases, Akt and GSK-3, which act on a Ser cluster in the same NHE3 C-terminal domain that binds ezrin, and 2) these kinases regulate the dynamic association between ezrin and NHE3 to affect basal NHE3 activity [9]. CaMKII is therefore generally inactive in the presence of basal levels of Ca 2+ , and active CaMKII is generally created via the autophosphorylation and release of the kinase sequence from the CaMKII autoinhibitory domain.…”

Section: Nhe3mentioning

confidence: 99%

“…In the nephron, individual NHE isoforms have different functions. These functions are reflected in their differential expression along the segments of the nephron, their localization in renal epithelial cells at the basolateral (e.g., isoform NHE1) or apical surface (e.g., isoform NHE3), and their activation in response to distinct agonists [4][5][6][7][8][9][10][11][12][13].…”

Section: Nhementioning

confidence: 99%

ANG II, ANG-(1-7), ALDO and AVP biphasic effects on Na+/H+ transport: the role of cellular calcium

Mello-Aires¹,

Dellova²,

Castelo‐Branco³

et al. 2017

Nephrol Renal Dis

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This article reviews the main body of knowledge regarding NHE1 and NHE3 exchangers and their interaction with Angiotensin II, Angiotensin-(1-7), Aldosterone and Arginine Vasopressin, particularly their renal actions. This work addresses the biphasic effects of different hormonal doses on NHE1 or NHE3 in proximal tubule in Wistar, SHR (hypertensives) and their control WKY (normotensives) rats or MDCK cells (which share similarities with the collecting duct). The hormones were applied alone, with their inhibitors or plus agents that change the [Ca 2+ ]i. The data are compatible with hormonal stimulation of these exchangers by increases of [Ca 2+ ]i in lower range, and inhibition at high [Ca 2+ ]i. In MDCK cells and Wistar rats, low doses of ANG II, ALDO or AVP stimulated the exchangers, while high doses inhibited them. ANG-(1-7), in Wistar or WKY rats has inverse, dose-dependent effects. In SHR rats, the biphasic effects of ANG-(1-7) were similar to the effects of ANG II, ALDO or AVP in Wistar rats. The interactions between these effects may represent a mechanism that regulates extracellular volume. In hypertensives animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated hypertension. Figure 6 shows a schematic model to describe these biphasic hormonal effects.

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AKT and GSK-3 Are Necessary for Direct Ezrin Binding to NHE3 as Part of a C-terminal Stimulatory Complex

Cited by 19 publications

References 50 publications

Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

Increased Electrophoretic Mobility of Long-Type GATA-6 Transcription Factor upon Substitution of Its PEST Sequence

ANG II, ANG-(1-7), ALDO and AVP biphasic effects on Na+/H+ transport: the role of cellular calcium

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