Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of ...eukaryotic translation initiation factor 2 at Ser-51 on the α subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2α, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2α, inhibition of protein synthesis, stress granule formation, and survival.
The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the α subunit of eukaryotic translation initiation factor 2 ...(eIF2α) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2α phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2α phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic β cells. The results demonstrate that regulation of translation through eIF2α phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.
A role for phosphoinositides in the endocytosis of muscarinic cholinergic receptors (mAChRs) has been investigated via inhibition
of the activity of phosphatidylinositol-4-kinase (PI4K). Pretreatment ...of SH-SY5Y neuroblastoma cells with micromolar concentrations
of wortmannin (WT), LY-294002, or phenylarsine oxide (PAO), three chemically distinct agents known to inhibit PI4K, resulted
in both an inhibition of agonist-induced endocytosis of mAChRs and a selective reduction in the 32 P-labeling of phosphatidylinositol-4-phosphate. PAO-mediated inhibition of both receptor endocytosis and phosphoinositide
synthesis could be fully reversed by inclusion of the bifunctional thiol 2,3-dimercaptopropanol. The requirement for phosphoinositide
synthesis in mAChR endocytosis was independent of a role for these lipids in the maintenance of the cytoskeleton because disruption
of the latter with cytochalasin D, ML-7, or colchicine failed to inhibit receptor internalization. Determination of PI4K activity
in subcellular fractions of SH-SY5Y cells indicated that enzyme activity in fractions enriched in endocytic vesicles and cytosol
was preferentially inhibited by WT, LY-294002, and PAO, a profile consistent with the subcellular distribution of the 110-kDa
β isoform of PI4K, as determined by Western blot analysis. Activity of PI4Kβ present in immunoprecipitated cell lysates was
inhibited >75% by inclusion of each of the three inhibitors. These results indicate that ongoing synthesis of phosphoinositides
is necessary for mAChR endocytosis and that the activity of a WT-sensitive form of PI4K, such as PI4Kβ, is required.
The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of ...experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70-80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K+ or acidification of the cytosol also resulted in > 80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K+, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca2+ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K+ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.
: The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of ...oxotremorine‐M to SH‐SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t1/2 = 2 min) and paxillin that were independent of integrin‐extracellular matrix interactions, cell attachment, and the production of phosphoinositide‐derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY‐294002, under conditions in which the synthesis of phosphatidylinositol 4‐phosphate was markedly attenuated. These results indicate that muscarinic receptor‐mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH‐SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.
: The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist‐induced internalization has been investigated. Incubation of SH‐SY5Y neuroblastoma cells with ...oxotremorine‐M resulted in a time‐dependent endocytosis of both muscarinic receptors and α subunits of Gq and G11, but not of isoforms of phosphoinositide‐specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist‐induced increases in diacylglycerol mass and in 32P‐phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist‐induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4‐kinase, none was enriched when compared with cell lysates. Both 32P‐labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5‐bisphosphate, whereas the synthesis of phosphatidylinositol 4‐phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5‐bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.
Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an ...increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t1/2 = 14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5-10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.
Fura-2 digital imaging microfluorimetry was used to evaluate the Ca2+ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate ...phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca2+i) but of different magnitudes (ET-1 = ATP > Oxo-M > NE). The Ca2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca2+ influx. The Ca2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca2+ signal, which was characteristic of the response to Oxo-M, was observed in < 40% of cells stimulated with ET-1 and absent from Ca2+ signals elicited after NE addition. Removal of extracellular Ca2+ enhanced the rate of decay of Ca2+ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca2+ influx. In the absence of extracellular Ca2+, NE elicited asynchronous multiple Ca2+ transients. In either the absence or presence of extracellular Ca2+-, > 94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were approximately 74 and approximately 48%.
To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (Gp), we ...have evaluated the contribution of Gp activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC50 of approximately 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC50 of approximately 3.5 microM). When added to digitonin-permeabilized cells, NaF or guanosine-5'-O-thiotriphosphate (GTP gamma S) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTP gamma S, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2-5 mM concentrations of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) fully blocked phosphoinositide hydrolysis elicited by GTP gamma S, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDP beta S resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTP gamma S and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 microM U-73122 (54-71%). The presence of either NaF or GTP gamma S also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of Gp.
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