Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gα_s plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.     

GABA(A) Receptor Pi (GABRP) Stimulates Basal-like Breast Cancer Cell Migration through Activation of Extracellular-regulated Kinase 1/2 (ERK1/2)

Breast cancer is a heterogeneous disease comprised of distinct subtypes predictive of patient outcome. Tumors of the basal-like subtype have a poor prognosis due to inherent aggressiveness and the lack of targeted therapeutics. Basal-like tumors typically lack estrogen receptor-α, progesterone receptor and HER2/ERBB2, or in other words they are triple negative (TN). Continued evaluation of basal-like breast cancer (BLBC) biology is essential to identify novel therapeutic targets. Expression of the pi subunit of the GABA(A) receptor (GABRP) is associated with the BLBC/TN subtype, and herein, we reveal its expression also correlates with metastases to the brain and poorer patient outcome. GABRP expression in breast cancer cell lines also demonstrates a significant correlation with the basal-like subtype suggesting that GABRP functions in the initiation and/or progression of basal-like tumors. To address this postulate, we stably silenced GABRP in two BLBC cell lines, HCC1187 and HCC70 cells. Decreased GABRP reduces in vitro tumorigenic potential and migration concurrent with alterations in the cytoskeleton, specifically diminished cellular protrusions and expression of the BLBC-associated cytokeratins, KRT5, KRT6B, KRT14, and KRT17. Silencing GABRP also decreases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) in both cell lines and selective inhibition of ERK1/2 similarly decreases the basal-like cytokeratins as well as migration. Combined, these data reveal a GABRP-ERK1/2-cytokeratin axis that maintains the migratory phenotype of basal-like breast cancer. GABRP is a component of a cell surface receptor, thus, these findings suggest that targeting this new signaling axis may have therapeutic potential in BLBC.     

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.     

Signaling to Extracellular Signal-regulated Kinase from ErbB1 Kinase and Protein Kinase C: FEEDBACK,HETEROGENEITY, AND GATING*

Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the “gradedness” or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a “gating” or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.     

The Lysosome Rupture-activated TAK1-JNK Pathway Regulates NLRP3 Inflammasome Activation

Lysosome rupture triggers NLRP3 inflammasome activation in macrophages. However, the underlying mechanism is not fully understood. Here we showed that the TAK1-JNK pathway, a MAPK signaling pathway, is activated through lysosome rupture and that this activation is necessary for the complete activation of the NLRP3 inflammasome through the oligomerization of an adapter protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). We also revealed that the activation of the TAK1-JNK pathway is sustained through Ca²⁺ ions and that calcium/calmodulin-dependent protein kinase type II functions upstream of the TAK1-JNK pathway and specifically regulates lysosome rupture-induced NLRP3 inflammasome activation. These data suggest a novel role for the TAK1-JNK pathway as a critical regulator of NLRP3 inflammasome activation.     

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

The vacuolar H⁺-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V₁ sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO₃⁻-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H⁺ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO₃⁻-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.     

Nuclear Calcium Signaling Induces Expression of the Synaptic Organizers Lrrtm1 and Lrrtm2

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2–4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca²⁺/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2.     

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na⁺, K⁺-ATPase is a cell–cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na⁺, K⁺-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.     

血清通过调节mGluR1介导的信号通路调控细胞的生长与凋亡

血清因子能调节细胞的生长与凋亡,但是其分子机制尚不清楚.通过细胞培养基中血清存在与否,研究了代谢型谷氨酸受体1(mGluR1)介导的胞外信号调节激酶(ERK),蛋白激酶B(PKB/AKT)通路的活化及其对细胞生长与凋亡的影响.在过量表达mGluR1的HEK293细胞中,血清饥饿促进了mGluR1对ERK,AKT信号通路的活化;细胞凋亡剂STS应激损伤时,受体激动剂DHPG可降低细胞活性,促进细胞凋亡.在大鼠胶质瘤细胞中,与过表达mGluR1的HEK293细胞的结果相反,血清有助于mGluR1对ERK,AKT通路的活化作用;STS应激损伤时,内源性mGluR1的活化抑制了细胞凋亡.结果表明:血清中存在的细胞因子,通过细胞中表达水平不同的mGluR1受体,调节受体介导的信号通路,从而调控细胞生长与凋亡.本文可能揭示了一种血清调节细胞生长与凋亡的新机制.     

Molecular Mechanisms of Calcium-sensing Receptor-mediated Calcium Signaling in the Modulation of Epithelial Ion Transport and Bicarbonate Secretion

Epithelial ion transport is mainly under the control of intracellular cAMP and Ca²⁺ signaling. Although the molecular mechanisms of cAMP-induced epithelial ion secretion are well defined, those induced by Ca²⁺ signaling remain poorly understood. Because calcium-sensing receptor (CaSR) activation results in an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) but a decrease in cAMP levels, it is a suitable receptor for elucidating the mechanisms of [Ca²⁺]_cyt-mediated epithelial ion transport and duodenal bicarbonate secretion (DBS). CaSR proteins have been detected in mouse duodenal mucosae and human intestinal epithelial cells. Spermine and Gd³⁺, two CaSR activators, markedly stimulated DBS without altering duodenal short circuit currents in wild-type mice but did not affect DBS and duodenal short circuit currents in cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice. Clotrimazole, a selective blocker of intermediate conductance Ca²⁺-activated K⁺ channels but not chromanol 293B, a selective blocker of cAMP-activated K⁺ channels (KCNQ1), significantly inhibited CaSR activator-induced DBS, which was similar in wild-type and KCNQ1 knockout mice. HCO₃⁻ fluxes across epithelial cells were activated by a CFTR activator, but blocked by a CFTR inhibitor. CaSR activators induced HCO₃⁻ fluxes, which were inhibited by a receptor-operated channel (ROC) blocker. Moreover, CaSR activators dose-dependently raised cellular [Ca²⁺]_cyt, which was abolished in Ca²⁺-free solutions and inhibited markedly by selective CaSR antagonist calhex 231, and ROC blocker in both animal and human intestinal epithelial cells. Taken together, CaSR activation triggers Ca²⁺-dependent DBS, likely through the ROC, intermediate conductance Ca²⁺-activated K⁺ channels, and CFTR channels. This study not only reveals that [Ca²⁺]_cyt signaling is critical to modulate DBS but also provides novel insights into the molecular mechanisms of CaSR-mediated Ca²⁺-induced DBS.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

Homer2 Protein Regulates Plasma Membrane Ca2+-ATPase-mediated Ca2+ Signaling in Mouse Parotid Gland Acinar Cells

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca²⁺-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca²⁺ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca²⁺ signaling in parotid gland acinar cells using Homer2-deficient (Homer2^−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca²⁺-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca²⁺-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca²⁺ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca²⁺ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca²⁺ signaling in parotid acinar cells.     

Cytosolic Branched Chain Aminotransferase (BCATc) Regulates mTORC1 Signaling and Glycolytic Metabolism in CD4+ T Cells

Here we show that expression of the cytosolic branched chain aminotransferase (BCATc) is triggered by the T cell receptor (TCR) of CD4⁺ T cells. Induction of BCATc correlates with increased Leu transamination, whereas T cells from the BCATc^−/− mouse exhibit lower Leu transamination and higher intracellular Leu concentrations than the cells from wild type (WT) mice. Induction of BCATc by TCR in WT cells is prevented by the calcineurin-nuclear factor of activated T cells (NFAT) inhibitor, cyclosporin A (CsA), suggesting that NFAT controls BCATc expression. Leu is a known activator of the mammalian target of rapamycin complex 1 (mTORC1). mTOR is emerging as a critical regulator of T cell activation, differentiation, and metabolism. Activated T cells from BCATc^−/− mice show increased phosphorylation of mTORC1 downstream targets, S6 and 4EBP-1, indicating higher mTORC1 activation than in T cells from WT mice. Furthermore, T cells from BCATc^−/− mice display higher rates of glycolysis, glycolytic capacity, and glycolytic reserve when compared with activated WT cells. These findings reveal BCATc as a novel regulator of T cell activation and metabolism and highlight the important role of Leu metabolism in T cells.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.     

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin^S66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.     

Excessive Na+/H+ Exchange in Disruption of Dendritic Na+ and Ca2+ Homeostasis and Mitochondrial Dysfunction following in Vitro Ischemia

Neuronal dendrites are vulnerable to injury under diverse pathological conditions. However, the underlying mechanisms for dendritic Na⁺ overload and the selective dendritic injury remain poorly understood. Our current study demonstrates that activation of NHE-1 (Na⁺/H⁺ exchanger isoform 1) in dendrites presents a major pathway for Na⁺ overload. Neuronal dendrites exhibited higher pH_i regulation rates than soma as a result of a larger surface area/volume ratio. Following a 2-h oxygen glucose deprivation and a 1-h reoxygenation, NHE-1 activity was increased by ∼70–200% in dendrites. This elevation depended on activation of p90 ribosomal S6 kinase. Moreover, stimulation of NHE-1 caused dendritic Na⁺_i accumulation, swelling, and a concurrent loss of Ca²⁺_i homeostasis. The Ca²⁺_i overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na⁺/Ca²⁺ exchange prevented these changes. Mitochondrial membrane potential in dendrites depolarized 40 min earlier than soma following oxygen glucose deprivation/reoxygenation. Blocking NHE-1 activity not only attenuated loss of dendritic mitochondrial membrane potential and mitochondrial Ca²⁺ homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na⁺ entry and subsequent Na⁺/Ca²⁺ exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.     

Loss of caveolin-1 impairs retinal function due to disturbance of subretinal microenvironment

Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.