Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

Nucleosome-associated protein kinases in murine erythroleukemia cells.

Chromatin subunits from murine erythroleukemia cells were prepared by a method which releases actively transcribing genes. Two casein kinase activities (CK₁ and CK₂) were isolated from these nucleosomes by gel nitration in 0.5 m NaCl. CK₁ (M_r ~ 200,000) and CK₂ (M_r ~ 35,000) were further purified by phosphocellulose chromatography and characterized with regard to several parameters which may regulate their activity in vivo. CK₁ has an NaCl optimum of 0.14 m, utilizes GTP as phosphate donor ~25% as efficiently as ATP, and phosphorylates a discrete group of high molecular weight nonhistone proteins in the unfractionated chromatin starting material. CK₂ has an NaCl optimum of 0.24 m, cannot utilize GTP, and modifies a different group of nonhistones. Both kinases are inhibited by concentrations of hemin (<50 μm) which efficiently induce globin gene expression in erythroleukemia cells. A histone kinase resolved during the gel filtration step is unaffected by hemin. An investigation of the mode of hemin inhibition reveals that CK₁ and CK₂ interact in different fashions with the inhibitor.     

Kinetic modelling of the effect of alpha subunit phosphorylation on the activity of the protein synthesis initiation factor eIF-2

The ability of eIF-2.GDP in which the alpha subunit of eIF-2 is phosphorylated (eIF-2(alpha P).GDP) to act as a competitive inhibitor of eIF-2B-catalysed exchange of eIF-2-bound GDP has been investigated by modelling data provided by Rowlands et al. (J. Biol. Chem. 263, 5526-5533:1988). Some revision of previously determined dissociation and rate constants proved to be necessary. Under the conditions employed it was not possible to demonstrate significant inhibition of GDP exchange by eIF-2 (alpha P).GDP without substantial increase in its affinity for eIF-2B over that of eIF-2.GDP. Classic double reciprocal plots for competitive inhibition were found only when [eIF-2B] was low in relation to [eIF-2 (alpha P).GDP]. Relatively high cellular [eIF-2B] lessens the inhibitory effect of eIF-2(alpha P).GDP and suggests the possibility of other potential controls of initiation.     

Purification of a novel eIF-2 alpha protein kinase from calf brain

A new eukaryotic initiation factor 2 kinase has been purified for the first time from calf brain cytosol. The purification of a nonabundant novel protein kinase activity, designated as PKI, that phosphorylates the alpha subunit of eukaryotic initiation factor 2 is described. The protein kinase activity was assayed using purified initiation factor 2 as a substrate and was purified by ammonium sulphate precipitation, conventional chromatography in heparin-Sepharose and phosphocellulose and by high performance size exclusion and anion exchange chromatographies. The protein kinase activity elutes in the region of 140,000 in the size exclusion chromatography and is associated with two different polypeptides a and b, with relative molecular masses of 38,000 and 20,000 and an approximate ratio of 2.5-3.0:1. The protein kinase does not phosphorylate casein or histones and it is independent of cyclic nucleotides. It can be classified as a serine kinase since the phosphorylation of the alpha subunit of eIF-2 is produced in serine residues. Under these conditions none of the kinase subunits are phosphorylated.     

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.     

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.     

Two phosphorylation sites on eIF-2 alpha

Protein synthesis in mammalian cells can be regulated through phosphorylation/dephosphorylation of the alpha subunit of initiation factor 2, eIF-2. Two specific kinases have been identified that apparently phosphorylate the same site(s). Controversy exists as to whether serine-48 is a phosphorylation site in addition to serine-51. A recent publication is discussed that, in this author's view, answers the question of the phosphorylation sites. It is suggested that phosphorylation proceeds sequentially with serine-51 being the first and serine-48 the second phosphorylation site. Phosphorylation of both sites is required for inhibition of protein synthesis.     

Specific phosphorylation of the beta subunit of eIF-2 factor from brain by three different protein kinases

The eukaryotic initiation factor 2 (eIF-2) from calf brain has been purified to homogeneity and free of endogenous kinase activity. Phosphorylation of eIF-2 factor has been examined with four different protein kinases. Casein kinase II, calcium/phospholipid-dependent protein kinase and cyclic AMP-dependent protein kinase from brain, phosphorylate the beta subunit of eIF-2, whilst hemin-controlled inhibitor phosphorylate the alpha subunit of the factor. According to the peptide maps obtained, the phosphorylation sites of the factor by the three beta kinases are specific and distinct. These data suggest a different regulation for the beta subunit through this modification.     

A kinase able to phosphorylate exogenous protein synthesis initiation factor eIF-2 alpha is present in lysates of mengovirus-infected L cells.

Infection of mouse L929 cells by mengovirus resulted in the expression of a kinase activity that selectively phosphorylated the small, 38,000-molecular-weight subunit of eucaryotic initiation factor 2 and histone H2. This kinase activity was independent of host cell RNA synthesis and was located in the postribosomal supernatant (S-100 fraction) early after infection (up to 3 h). At later times after infection (5 h), kinase activity was also associated with the polysome fraction. The kinase present in the S-100 fraction bound strongly to DEAE-cellulose, its peak activity eluting at 0.5 M KCl. Kinase activity was independent of the presence of exogenous double-stranded RNA, and KCl at concentrations greater than 0.1 M inhibited eucaryotic initiation factor 2 phosphorylation. The 67,000-molecular-weight phosphoprotein activated in interferon-treated cells by double-stranded RNA was not detected by standard phosphorylation assays in lysates from mengovirus-infected cells. Labeling of this protein in vivo during 5 h of infection was also not detected. The DEAE-cellulose-purified mengovirus kinase inhibited protein synthesis in reticulocyte lysates, and the inhibition was not reversible by high concentrations of poly(I).poly(C).     

Specific phosphorylation of pig liver initiation factor eIF-2 by the N-ethylmaleimide-treated hemin-controlled translational inhibitor

The specific phosphorylation of pig liver initiation factor 2(eIF-2) by the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) from rabbit reticulocytes was investigated. The inhibitor phosphorylated the serine residue of the alpha subunit of eIF-2 (eIF-2 alpha) and 1 mol of phosphate was incorporated into 1 mol of eIF-2 alpha by the inhibitor on maximal phosphorylation, even when eIF-2 was pretreated with alkaline phosphatase prior to phosphorylation. The 32P-labeled eIF-2 alpha was subjected to tryptic digestion and the tryptic digest was analyzed by two-dimensional peptide mapping on a cellulose thin-layer sheet. After 94 h digestion, the autoradiograph of the peptide map showed a single 32P-labeled band with a molecular weight of approximately 1,200. These findings suggest that one specific serine residue of pig liver eIF-2 alpha was phosphorylated by the NEM-treated HCI.     

Differential effect of Mn2+ on the hemin-controlled translational repressor and the double-stranded RNA-activated inhibitor

The inhibition of protein synthesis that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin is due to the activation of a protein kinase termed the hemin-controlled translational repressor, and that occurring when reticulocyte lysate is incubated with a low level of double-stranded RNA is mediated by the activation of a separate protein kinase termed the double-stranded RNA-activated inhibitor. Both the hemin-controlled translational repressor and the double-stranded RNA-activated inhibitor act by phosphorylating the M_r = 35 000 (α) subunit of eIF-2. MnCl₂ (0.5 mM) partly reverses the inhibition of protein synthesis produced by hemin deficiency but not that induced by double-stranded RNA. In addition, Mn²⁺ reverses the inhibition of binding of [³⁵S]Met-tRNA_f to reticulocyte ribosomal components, isolated on Sepharose 6B, produced by the hemin-controlled translational repressor but not by the double-stranded RNA-activated inhibitor. The effect of Mn²⁺ is mediated at the level of activation and eIF-2α kinase activity of these two regulatory protein kinases. Specifically, Mn²⁺ inhibits activation of the hemin-controlled translational repressor in the absence of hemin and the phosphorylation of eIF-2α by pre-activated translational repressor. In contrast, the phosphorylation of eIF-2α by the double-stranded RNA-activated inhibitor is not suppressed by Mn²⁺, and the activation and autophosphorylation of this inhibitor is enhanced by Mn²⁺. Finally, while the activation and inactivation of the hemin-controlled translational repressor does not appear to be mediated by autophosphorylation and dephosphorylation, the activation of the double-stranded RNA-activated inhibitor does appear to require autophosphorylation.     

Thromboxane A2 synthesis in human erythroleukemia cells

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.     

Evaluation and significance of kinetic parameters governing function of protein synthesis initiation factors eIF-2 and eIF-2B

Published data dealing with the formation of the ternary complex eIF-2 X GTP X met-tRNAi involved in eukaryotic initiation have been evaluated to calculate the expected inhibition by GDP and the role of eIF-2B in limiting this inhibition. It is concluded that cellular levels of GDP are unlikely seriously to inhibit ternary complex formation if the reaction can proceed to equilibrium. However, derivation of 'on' and 'off' rates for the interaction of GTP and GDP with eIF-2 demonstrates that these are too slow in the absence of eIF-2B to support active protein synthesis, particularly if eIF-2 is released from ribosomes as eIF-2 X GDP. Whilst eIF-2 X GDP and eIF-2 X GTP appear to dissociate equally slowly, it is concluded that GDP binds to eIF-2 100-times faster than GTP. Addition of eIF-2B has the effect of raising k-1 for both GDP and GTP several hundred-fold and k+1 50- and 7000-fold, respectively. Thus, a kinetic block can be relieved even if there is no change in the thermodynamic state. Phosphorylation of the alpha-subunit of eIF-2 appears to affect only those parameters influenced by eIF-2B. The reported rescue of inhibited lysates by addition of 1 mM GTP is not by mass action but by some other mechanism. Consideration of the kinetic parameters favours the formation of a ternary complex of eIF-2 X eIF-2B X GDP en route to eIF-2 X GTP as opposed to displacement of GDP from eIF-2 X GDP by eIF-2B.     

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini.     

Tyrphostin-induced differentiation of mouse erythroleukemia cells

Inhibitors of protein-tyrosine kinases (TPKs) from the tyrphostins family induce terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. The most potent tyrphostin was found to be AG-568 which was therefore investigated in more detail. Just prior to differentiation the inhibition of tyrosine phosphorylation of a pp⁹⁷ protein band was noted. We also found that AG-568 treatment induces the appearance of a putative differentiation factor which could induce tyrphostin-independent differentiation in untreated cells. Our study suggests that the inhibition of tyrosine phosphorylation by AG-568 leads to the production of differentiating factor(s) which induce the MEL cells to differentiate.