Novel Phorbol Ester-binding Motif Mediates Hormonal Activation of Na+/H+ Exchanger

Protein kinase C (PKC) is considered crucial for hormonal Na⁺/H⁺ exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.     

Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3′-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3′-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.     

Calmodulin kinase II constitutively binds, phosphorylates, and inhibits brush border Na+/H+ exchanger 3 (NHE3) by a NHERF2 protein-dependent process

The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca(2+)-dependent manner, with less association when Ca(2+) is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca(2+). CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na(+)-absorptive cell.     

CK2 phosphorylation of an acidic Ser/Thr di-isoleucine motif in the Na+/H+ exchanger NHE5 isoform promotes association with beta-arrestin2 and endocytosis

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins β-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by β-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to β-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of β-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of β-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with β-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to β-arrestin2 binding and trafficking along the recycling endosomal pathway.     

Identification of a Novel HSP70-binding Cochaperone Critical to HSP90-mediated Activation of Small Serine/Threonine Kinase

We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (K_d ∼10 nm), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK.     

Intracellular pH Increase Driven by an Na+/H+ Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pH_i) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pH_iof 0.3 pH unit after activation by excess K⁺. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pH_iwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K⁺induced GVBD but the pH_iof K⁺-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pH_iincreases in K⁺-activated oocytes were otherwise proportional to the external Na⁺concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K⁺-induced increase of pH_ibut did not block GVBD. These two derivatives were able, however, to retard K⁺-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K⁺-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pH_iincrease, driven by a typical Na⁺/H⁺exchanger, follows activation of surf clam oocytes but that this pH_iincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

A hallmark feature of Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca²⁺-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active (∼70–100%), implicating that it is no longer regulated and that its dramatically increased Ca²⁺/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15–25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca²⁺/CaM, both in vitro and within cells. Altered K_m and V_max made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca²⁺ signals, but prevents complete uncoupling from subsequent cellular stimulation.     

Keeping It Simple,Transport Mechanism and pH Regulation in Na+/H+ Exchangers

Na⁺/H⁺ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na⁺/H⁺ exchangers, where a single binding site is alternatively occupied by Na⁺ or one or two H⁺ ions. The proposed transport mechanism inherently down-regulates Na⁺/H⁺ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na⁺/H⁺ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na⁺/H⁺ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na⁺/H⁺ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na⁺/H⁺ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na⁺/H⁺ exchangers.     

Reciprocal Negative Regulation of PDK1 and ASK1 Signaling by Direct Interaction and Phosphorylation

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H₂O₂, tumor necrosis factor α, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser⁹⁶⁷, a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H₂O₂-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser³⁹⁴ and Ser³⁹⁸, indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.     

p38 Regulates Pigmentation via Proteasomal Degradation of Tyrosinase

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by α-melanocyte-stimulating hormone (α-MSH) and ultraviolet irradiation. Previous studies have shown that α-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and α-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

B-Raf associates with and activates the NHE1 isoform of the Na+/H+ exchanger

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.     

Phosphorylation of a Novel Cytoskeletal Protein (RsmP) Regulates Rod-shaped Morphology in Corynebacterium glutamicum

Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.     

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr¹⁰⁹ in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr¹⁰⁹ by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/−) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.     

GSK-3 Represses Growth Factor-inducible Genes by Inhibiting NF-��B in Quiescent Cells

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-κB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IκB kinase, leading to phosphorylation and degradation of IκBα and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-κB through inhibition of IκB kinase. This inhibition of NF-κB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.     

Activation of Na+/H+ Exchanger NHE3 by Angiotensin II Is Mediated by Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3 (IRBIT) and Ca2+/Calmodulin-dependent Protein Kinase II

Angiotensin II (ANG II) stimulates renal tubular reabsorption of NaCl by targeting Na⁺/H⁺ exchanger NHE3. We have shown previously that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) plays a critical role in stimulation of NHE3 in response to elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i). In this study, we investigated the role of IRBIT in mediating NHE3 activation by ANG II. IRBIT is abundantly expressed in the proximal tubules where NHE3 is located. ANG II at physiological concentrations stimulates NHE3 transport activity in a model proximal tubule cell line. ANG II-induced activation of NHE3 was abrogated by knockdown of IRBIT, whereas overexpression of IRBIT enhanced the effect of ANG II on NHE3. ANG II transiently increased binding of IRBIT to NHE3 at 5 min but became dissociated by 45 min. In comparison, it took at least 15 min of ANG II treatment for an increase in NHE3 activity and NHE3 surface expression. The stimulation of NHE3 by ANG II was dependent on changes in [Ca²⁺]_i and Ca²⁺/calmodulin-dependent protein kinases II. Inhibition of CaMKII completely blocked the ANG II-induced binding of IRBIT to NHE3 and the increase in NHE3 surface abundance. Several serine residues of IRBIT are thought to be important for IRBIT binding. Mutations of Ser-68, Ser-71, and Ser-74 of IRBIT decreased binding of IRBIT to NHE3 and its effect on NHE3 activity. In conclusion, our current findings demonstrate that IRBIT is critically involved in mediating activation of NHE3 by ANG II via a Ca²⁺/calmodulin-dependent protein kinases II-dependent pathway.     

Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H⁺-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na⁺/H⁺ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.