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Reconsolidation of a context long-term memory in the terrestrial snail requires protein synthesis.

We investigated the influence of the protein synthesis blocker anisomycin on contextual memory in the terrestrial snail Helix. Prior to the training session, the behavioral responses in two contexts were similar. Two days after a session of electric shocks (5 d) in one context only, the context conditioning was observed as the significant difference of behavioral response amplitudes in two contexts. On the day following testing of context learning, a session of "reminding" was performed, immediately after which the snails were injected with anisomycin or vehicle. Testing of long-term context memory has shown that only anisomycin injections impaired the context conditioning. In control series, the snails were injected after the training session with anisomycin/saline without reminding, and no impairment of the long-term context memory was observed, while injection of anisomycin during the training session completely abolished the long-term memory. No effects of anisomycin on the short-term memory were observed. Surprisingly, injection of anisomycin after the reminding combined with reinforcing stimuli elicited no effect on the context memory. Differences between single-trial and multisession learning are discussed.

Animals↗

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization.

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.

Anisomycin↗

Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK.

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.

Animals↗

Circadian rhythm from the eye of Aplysia: temperature compensation of the effects of protein synthesis inhibitors.

1. The circadian rhythm of compound action potentials (CAP) frequency recorded from the isolated eye of Aplysia in culture medium and darkness was subjected to step and pulse treatments with anisomycin, a protein synthesis inhibitor. 2. The step application of anisomycin and its continued presence in the culture medium lengthened the period of the rhythm in a dose-dependent manner. At 10(-8) M the period was increased from the normal 26.5 h to about 28 h and at 10(-7) M the period was lengthened to 31 h or longer. At 10(-6) M the rhythm was suppressed but the CAP activity continued without the cyclic variations in CAP frequency. 3. Six-hour pulses of anisomycin at 10(-6) M caused phase-dependent phase-shifts of the rhythm. Maximum phase delays of 15 h were obtained at CT (circadian time) 2 and maximum phase advances of 4 h were obtained at CT 6. The phase response curves at 12, 15 and 17 degrees C were essentially identical. 4. Anisomycin appears to act rather selectively on the circadian clock mechanism. It does not alter the CAP amplitude and duration and it does not alter the bursting pacemaker mechanism of the optic nerve CAP or central neurones. 5. The results support the hypothesis that the synthesis of a protein or polypeptide on eucaryotic ribosomes is an essential part of the circadian clock timing mechanism. The sensitivity of the clock to anisomycin is the same at three different temperatures (12, 15 and 17 degrees C) within the physiological range of temperatures for Aplysia, as expected for a clock whose period length is temperature compensated (Q10 1.02) over that same range. 6. At the critical phases of CT 1-4, anisomycin pulses often caused unusual perturbations of the rhythm. These effects are consistent with the hypothesis that the circadian rhythm is a multioscillator system.

Action Potentials↗

Selective stimulation of Hsp27 and alphaB-crystallin but not Hsp70 expression by p38 MAP kinase activation.

The levels of Hsp27 and alphaB-crystallin in C6 rat glioma cells, that had been heated at 43 degrees C for 30 min with a subsequent culture for 16 h at 37 degrees C, were markedly increased. The exposure of the cells to a low concentration (0.1-3 microg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and alphaB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and alphaB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1-3 microg/ml anisomycin. The results of a reporter assay, using an alphaB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of alphaB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 microg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO2 or CdCl2. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 microg/ml of anisomycin. The heat-induced accumulations of Hsp27 and alphaB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and alphaB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and alphaB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.

Animals↗

Protein synthesis inhibition blocks the induction of mossy fiber long-term potentiation in vivo.

Protein synthesis inhibitors block the maintenance of NMDA receptor-dependent long-term potentiation (LTP) both in vivo and in vitro. Protein synthesis inhibitors block mossy fiber(MF) LTP maintenance in vitro, but little is known about the effect of protein synthesis inhibitors on either induction or maintenance in MF-LTP in vivo. Here we study the role of protein synthesis in the induction of long-term potentiation at the mossy fiber-CA3 hippocampal synapse in vivo in anesthetized rats. The protein synthesis inhibitor anisomycin was administered at different doses (0.04, 10, or 40 nmol) into area CA3 15 min before delivering high-frequency stimulation (two times at 100 Hz, 1 sec). Anisomycin blocked MF-LTP induction in a dose-dependent manner; both 40 and 10 nmol blocked MF-LTP induction, but a lower dose of 0.04 nmol was without effect. The inhibitory effect of anisomycin on protein synthesis was determined by measuring the incorporation of [(35)S]methionine into the newly synthesized proteins. Percentages of protein synthesis inhibition were determined by comparing [(35)S] incorporation of anisomycin-treated samples with vehicle controls. Doses of 0.04, 10, or 40 nmol of anisomycin produced 21, 82, or 83% inhibition of [(35)S]methionine incorporation, respectively. The effect of anisomycin was verified using a single dose of the protein synthesis inhibitor cycloheximide (40 nmol). Cycloheximide also blocked MF-LTP induction. These results suggest that protein synthesis plays an important role in the induction of mossy fiber long-term potentiation in vivo.

Action Potentials↗

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.

3T3 Cells↗

Sequential inhibition of sexual receptivity by progesterone is prevented by a protein synthesis inhibitor and is not causally related to decreased levels of hypothalamic progestin receptors in the female rat.

In the estrogen-primed ovariectomized female rat, the administration of progesterone facilitates sexual receptivity and activates proceptivity for many hours. If the progesterone dose is large (e.g., 2.5 mg), a refractory period follows, during which time animals are less sensitive to additional progesterone. This refractory period has been termed "sequential inhibition" and has been correlated previously with decreased levels of cytosol progestin receptors in the mediobasal hypothalamus-preoptic area (MBH-POA). Our present results indicate that the "sequential inhibition" of mating behavior by progesterone appears to involve a protein synthetic step that is not related causally to decreased levels of cytosol progestin receptors in the MBH-POA. Animals which received subcutaneous injections of the protein synthesis inhibitor, anisomycin (100 mg/kg in saline), 15 min prior to and again 3 hr after progesterone (2.5 mg in propylene glycol) treatment, displayed greater sexual receptivity at 24 hr than did animals which received subcutaneous saline injections. However, cytosol progestin receptor levels in the MBH-POA of animals which received anisomycin plus progesterone were decreased at 24 hr, relative to animals which received saline plus progesterone. Anisomycin did not produce positive or negative behavioral effects at 24 hr when administered 15 min prior to and again 3 hr after propylene glycol, suggesting that the behavioral effects of anisomycin appear to be related to selective blockade of progesterone action. In separate experiments, we administered single subcutaneous injections of anisomycin at various times relative to progesterone (2.5 mg) treatment. According to the time course of effective anisomycin application, the presumed protein synthesis which is responsible for the inhibition of mating behavior occurs between 3 and 13 hr after the administration of progesterone.

Animals↗

Cardioprotection by St Thomas' solution is mediated by protein kinase C and tyrosine kinase.

BACKGROUND: Intracellular signaling pathways, specifically the activation of protein kinase C and tyrosine kinase, are essential to the cardioprotection of ischemic preconditioning. We proposed that activation of PKC and TK contribute to the myocardial protection of St. Thomas' No. 2 cardioplegia solution (STC). MATERIALS AND METHODS: Isolated rat hearts were exposed to 40 min of global ischemia followed by 120 min of reperfusion. Before ischemia, hearts received no treatment (control; n = 7), STC (n = 7), phorbol 12-myristate 13-acetate (PMA; n = 6), PMA + chelerythrine (n = 6), anisomycin (n = 6), anisomycin + genistein (n = 7), STC + chelerythrine (n = 7), STC + genistein (n = 7), PMA + genistein (n = 7) or anisomycin + chelerythrine (n = 7). Left ventricular developed pressure (LVDP) recovery, myocardial infarct size, and lactate dehydrogenase release were measured. RESULTS: STC as well as PMA (protein kinase C activator) and anisomycin (tyrosine kinase activator) significantly reduced infarct size (6.9 +/- 2.9%, 9.6 +/- 2.1%, 14.0 +/- 4.4%) compared with controls (42.4 +/- 2.9%, P < 0.05). The infarct reduction of PMA and anisomycin were blocked by their inhibitors chelerythrine and genistein, respectively. Both chelerythrine (29.2 +/- 4.1%, P < 0.05) and genistein (40.4 +/- 4.3%, P < 0.05) attenuated the reduction of infarct size provided by STC. The recovery of LVDP improved with STC, PMA and anisomycin (72.6 +/- 1.4%, 60.4 +/- 4.7%, 57.2 +/- 4.6%) compared with control (33.8 +/- 3.6%, P < 0.05). Addition of chelerythrine or genistein to STC impaired recovery of LVDP (52.3 +/- 4.4%, 35.1 +/- 2.5%, P < 0.05) compared with STC treatment. CONCLUSION: Administration of the pharmacologic inhibitors chelerythrine and genistein blunts the cardioprotection caused by STC treatment.

Animals↗

p38 MAPK activation elevates serotonin transport activity via a trafficking-independent, protein phosphatase 2A-dependent process.

Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.

Animals↗

New targets for antivirals: the ribosomal A-site and the factors that interact with it.

Many viruses use programmed -1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins. Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation. Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited -1 frameshifting and the ability of yeast cells to propagate the L-A and M(1) dsRNA viruses (J. D. Dinman, M. J. Ruiz-Echevarria, K. Czaplinski, and S. W. Peltz, 1997, Proc. Natl. Acad. Sci. USA 94, 6606-6611). Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R. E. Schwartz, J. Liesch, O. Hensens, L. Zitano, S. Honeycutt, G. Garrity, R. A. Fromtling, J. Onishi, and R. Monaghan, 1988. J. Antibiot. (Tokyo) 41, 1774--1779), also inhibits -1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin. Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome. Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed -1 ribosomal frameshifting efficiencies and killer virus phenotypes. Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor. These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds.

Anisomycin↗

The role of protein synthesis in the hypothalamic mechanism mediating pyrogen fever.

The effects of inhibition of protein synthesis by anisomycin on the pathogenesis of fever and normal thermoregulatory processes were investigated in the conscious and unrestrained cat. Subcutaneous administration of 5.0-25.0 mg/kg of anisomycin prevented the fever normally evoked by an intravenous infusion of either 1.0 ml (10(8) organisms) of a 1:10 dilution of S. typhosa or 1.0-5.0 ml (3.5 x 10(5)-2.1 X 10(7) cells/ml) of endogenous pyrogen. In addition, systemic pre-treatment with anisomycin delayed and/or blocked the fever typically elicited by a direct micro-injection into the anterior hypothalamic, preoptic area (AH/POA) at AP 12.5-16.0 of 1.0 microliters of the endotoxin. Anisomycin did not alter the hyperthermic response to an anterior hypothalamic injection of either 1.0-7.0 micrograms/1.0 microliters of serotonin (5-HT) or 100.0 ng/1.0 microliters of prostaglandin (PGE). Inhibition of protein synthesis, furthermore, did not prevent the fall in body temperature usually produced by an intrahypothalamic micro-injection of 2.33-14.0 micrograms/1.0 microliters of either norepinephrine (NE) or dopamine (DA). The thermoregulatory capacity of the cat was unaffected by the administration of comparable doses of anisomycin, i.e., the animal was able to maintain normal body temperature (+/- 0.5 degrees C) when exposed to an ambient temperature of either 10 degrees C or 34 degrees C. These results strongly suggest that the synthesis of new protein within the region of the AH/POA is a functional requisite for the development of a pyrogen-induced fever.

Animals↗

Regulation of corticotropin-releasing factor receptor type 2beta mRNA by mitogen-activated protein kinases in aortic smooth muscle cells.

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors. CRF receptor type 2beta (CRFR2beta) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2beta mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2beta mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2beta mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2beta mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2beta mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2beta mRNA levels indirectly as a consequence of blocking protein synthesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and -insensitive pathways.

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38delta) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360-4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370-4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-alpha (TNF-alpha) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-alpha and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their "upstream" activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38delta in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-alpha or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-alpha inhibit eEF2 kinase via the phosphorylation of Ser-359.

Amino Acid Sequence↗

Paracrine neuroprotective effect of nitric oxide in the developing retina.

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.

Animals↗

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells.

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells.

Animals↗

Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

Anisomycin↗

Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.

Animals↗