Charles O. Brostrom

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: Identification of phospholipids as calcium-independent activators☆

Abstract Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca 2+ and a Ca 2+ -binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca 2+ and the Ca 2+ -dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations. The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca 2+ ·CDR complex. Both Ca 2+ ·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μ m with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 m m with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K m for each nucleotide remained unchanged while the V increased. The apparent K d for Mg 2+ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μ m and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca 2+ ·CDR and lysophosphatidyl choline activation of the enzyme.

Calcium-binding phosphoprotein from pig brain: Identification as a calcium-dependent regulator of brain cyclic nucleotide phosphodiesterase

Abstract A homogeneous, acidic, Ca 2+ -binding phosphoprotein from pig brain has been identified as the mediator of a Ca 2+ -dependent activation of a partially purified pig brain cyclic nucleotide phosphodiesterase. Crude extracts prepared from porcine brain have been resolved into two fractions by column chromatography with ECTEOLA-cellulose. One fraction possesses phosphodiesterase activity which is stimulated from 4- to 12-fold, depending upon the preparation and the assay conditions, by nanogram quantities of a purified Ca 2+ -binding phosphoprotein from pig brain at 5 × 10 −5 , m Ca 2+ . The stimulation is eliminated by ethylene glycol-bis(β-aminoethyl ether)- N,N′ -tetraacetate. The second fraction contains an endogenous, Ca 2+ -dependent activator of the phosphodiesterase activity. Further purification of the crude activator by hydroyxlapatite chromatography, Sephadex G-75 gel filtration, and acrylamide gel electrophoresis has established that the activator and the purified Ca 2+ -binding phosphoprotein are identical with respect to Chromatographic behavior, molecular weight, and electrophoretic migration. Both proteins bound Ca 2+ and were stable to boiling for 5 min. At saturating concentrations of the Ca 2+ -dependent regulator, half-maximal stimulation of the phosphodiesterase was observed at a Ca 2+ concentration of 4 × 10 −6 , m . Purified preparations of the acidic Ca 2+ -binding phosphoprotein from bovine adrenal medulla and testis were also active in stimulating the brain phosphodiesterase. Preparations of other known Ca 2+ -binding proteins, such as rabbit muscle troponin and pig brain S-100 protein, were nonstimulatory.

An improved protein binding assay for cyclic AMP

Abstract A simplified and inexpensive protein binding assay for cAMP has been developed for the rapid measurement of subpicomole quantities of the nucleotide. Conditions of assay were established in which interferences from salts and nucleotides present in biological samples were eliminated. Sodium chloride was demonstrated to reduce cAMP binding by denaturing the regulatory subunit of the protein kinase.

Inhibition of Translational Initiation by Activators of the Glucose-regulated Stress Protein and Heat Shock Protein Stress Response Systems ROLE OF THE INTERFERON-INDUCIBLE DOUBLE-STRANDED RNA-ACTIVATED EUKARYOTIC INITIATION FACTOR 2α KINASE

Depletion of endoplasmic reticulum (ER) Ca2+ perturbs protein folding and processing within the organelle while inhibiting translational initiation through activation of the double-stranded RNA-activated eukaryotic initiation factor (eIF)-2α kinase (PKR) (Prostko, C. R., Dholakia, J. N., Brostrom, M. A., and Brostrom, C. O. (1995) J. Biol. Chem. 270, 6211-6215). The glucose-regulated stress protein (GRP) chaperones are subsequently induced. We now report that sodium arsenite, a prototype for stressors fostering cytoplasmic protein misfolding, also inhibits translational initiation through activation of PKR while subsequently inducing the heat shock protein (HSP) chaperones. Arsenite neither mobilized ER-associated Ca2+ nor slowed peptide chain elongation. Various HSP-inducing chemicals caused rapid phosphorylation of eIF-2α. When incubated with double-stranded RNA, extracts derived from arsenite-treated cells displayed greater degrees of phosphorylation of PKR and eIF-2α than did control extracts. Cells overexpressing a dominant negative PKR mutation resisted translational inhibition and eIF-2α phosphorylation in response to ER or cytoplasmic stressors. Induction of either the HSP or GRP chaperones was accompanied by development of translational tolerance to either Ca2+-mobilizing agents or arsenite. Following induction of the HSPs by arsenite, cells remained susceptible to induction of the GRPs by Ca2+-mobilizing agents. Conversely, cells possessing induced GRP contents in response to Ca2+-mobilizing agents readily induced the HSPs in response to arsenite. It is concluded that the two chaperone systems function independently except for their mutual suppression of PKR.

The Dynamic Role of GRP78/BiP in the Coordination of mRNA Translation with Protein Processing

The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2α phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.

Reversible phosphorylation of eukaryotic initiation factor 2α in response to endoplasmic reticular signaling

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2α (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostkoet al. J. Biol. Chem. 267: 16751–16754, 1992). Alterations in eIF-2α phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2α in a manner blocked by antinomycin D. Phosphorylation of eIF-2α in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2α phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2α occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2α and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2α in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2α phosphorylation and translational repression.

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

Abstract The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca2+-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μ m GTP and 3.5-fold by 100 μ m Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca2+ concentrations; both phases of this response occurred at lower free Ca2+ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells.

Abstract A Ca 2+ -dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca 2+ -dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca 2+ -binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca 2+ and CDR. In subcellular fractionation experiments both the CDR and the Ca 2+ -dependent phosphodiesterase were predominantly located in the 100,000 g supernatant fraction. The apparent K m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μ m , respectively, when CDR was not rate limiting. Minor increases in the apparent K m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μ m Ca 2+ for the hydrolysis of 25 μ m cGMP and by 8 μ m Ca 2+ for the hydrolysis of 25 μ m cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca 2+ . The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.

Vasopressin-induced hypertrophy in H9c2 heart-derived myocytes

Protein synthesis in H9c2 heart-derived myocytes responds biphasically to arginine vasopressin (1 microM). An initial 50% inhibition attributable to Ca(2+) mobilization from the sarcoplasmic/endoplasmic reticulum is followed by a recovery that subsequently converts to a 1.5-fold stimulation. This study was undertaken to ascertain whether vasopressin programs H9c2 cells to undergo hypertrophy or to proliferate and whether early translational inhibition is required for programming. Translational suppression was observed only at vasopressin concentrations (>1 nM) causing extensive (>50%) depletion of Ca(2+) stores and was diminished at supraphysiologic extracellular Ca(2+) concentrations. Stimulation of protein synthesis, by contrast, was unaffected by changes in extracellular Ca(2+), depended on gene transcription, was suppressed by a protein kinase C pseudosubstrate sequence (peptide 19-27), and was observed at pM vasopressin concentrations. Activation of MAP kinases, phosphoinositide 3-kinase, calcineurin, S6 kinase, or eIF4 could not be implicated in the stimulation, which persisted for 24 h. Vasopressin-treated H9c2 cells underwent hypertrophy by standard criteria. Cellular protein accumulation occurred at pM hormone concentrations, was blocked by peptide 19-27, was observed regardless of retinoic acid pretreatment to prevent myogenic transdifferentiation, and preceded full repletion of Ca(2+) stores. It is proposed that H9c2 cells, which possess all basic features of V1-vasopressin receptor signaling, provide a convenient model for investigating vasopressin-induced myocyte hypertrophy. Early translational suppression is not needed for vasopressin-induced H9c2 myocyte hypertrophy whereas activation of protein kinase C appears essential.

Regulated expression of GRP78 during vasopressin‐induced hypertrophy of heart‐derived myocytes

Although the development of cellular hypertrophy is widely believed to involve Ca2+ signaling, potential supporting roles for sequestered Ca2+ in this process have not been explored. H9c2 cardiomyocytes respond to arginine vasopressin with an initial mobilization of Ca2+ stores and reduced rates of mRNA translation followed by repletion of Ca2+ stores, up‐regulation of translation beyond initial rates, and the development of hypertrophy. Rates of synthesis of the endoplasmic reticulum (ER) chaperones, GRP78 and GRP94, were found to increase preferentially at early times of vasopressin treatment. Total GRP78 content increased 2‐ to 3‐fold within 8 h after which the chaperone was subject to post‐translational modification. Preferential synthesis of GRP78 and the increase in chaperone content both occurred at pM vasopressin concentrations and were abolished at supraphysiologic Ca2+ concentrations. Co‐treatment with phorbol myristate acetate decreased vasopressin‐dependent Ca2+ mobilization and slowed appearance of new GRP78 molecules in response to the hormone, whereas 24 h pretreatment with phorbol ester prolonged vasopressin‐dependent Ca2+ mobilization and further increased rates of GRP78 synthesis in response to the hormone. Findings did not support a role for newly synthesized GRP78 in translational up‐regulation by vasopressin. However up‐regulation, which does not depend on Ca2+ sequestration, appeared to expedite chaperone expression. This report provides the first evidence that a Ca2+‐mobilizing hormone at physiologic concentrations signals increased expression of GRP78. Translational tolerance to depletion of ER Ca2+ stores, typifying a robust ER stress response, did not accompany vasopressin‐induced hypertrophy. J. Cell. Biochem. 83: 204–217, 2001.