The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca²⁺/CaM binds eEF-2K with high affinity (K_d(CaM)^app = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10⁴-fold (k_auto = 2.6 ± 0.3 s⁻¹). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca²⁺/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k_cat^app for peptide substrate phosphorylation by 10³-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k_cat^app/K_m(Pep-S)^app), with equal contributions from k_cat^app and K_m(Pep-S)^app, suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.     

Taurine Inhibits Protein Kinase C-Catalyzed Phosphorylation of Specific Proteins in a Rat Cortical P2 Fraction

Abstract: We previously reported that taurine inhibits the phosphorylation of specific proteins in a P₂ synaptosomal fraction prepared from the rat cortex. In the present study, the regulation of the phosphorylation of an ～20K M_r protein whose phosphorylation is inhibited by taurine was further investigated. The phosphorylation of the ～20K M_r protein in a hypo-osmotically shocked P₂ fraction from rat cortex was dependent on the free Ca²⁺ in the reaction medium. Depolarization induced by 30 mM K⁺ stimulated the phosphorylation of the ～20K M_r protein in an intact synaptosomal P₂ preparation by 30-fold. This stimulation was inhibited 35% by taurine, whereas guanidinoethanesulfonic acid, a taurine analogue, did not have any effect, thereby indicating the specificity of taurine. Addition of phorbol 12-myristate 13-acetate, a phorbol ester, together with phosphatidylserine, stimulated the phosphorylation of the ～20K M_r protein in the hypo-osmotically shocked P₂ synaptosomal fraction by fivefold, whereas cyclic AMP, cyclic GMP, and calmodulin did not have any effect on the phosphorylation of this particular protein. Phorbol 12-myristate 13-acetate–stimulated phosphorylation of the ～20K M_r protein is blocked 30% by taurine. Taurine also inhibited phorbol 12-myristate 13-acetate-activated phosphorylation of two other proteins that were similar in molecular weight and isoelectric point to the ～20K M_r protein on two-dimensional gels. These results suggest that taurine modulates the phosphorylation of specific proteins regulated by the signal transduction system in the brain. Thus, taurine may modulate neuroactivity by inhibiting the phosphorylation of specific proteins involved in regulatory function.     

Involvement of a dithiol protein in mitochondrial energy-linked functions and its relation to coupling factor B.

The coupling factor protein isolated previously in pure form with a molecular weight of 11–12 × 10³ (K.-S. You and Y. Hatefi, 1976, Biochim. Biophys. Acta423, 398–412) has been shown to restore ATP-induced NAD reduction by succinate, transhydrogenation from NADH to NADP, and ATP-³³P_i exchange to submitochondrial particles rendered deficient by extraction with 1 m NH₄OH. The factor also stimulated the oxidative phosphorylation activity of the extracted particles 2.5- to >3-fold. The stimulatory effect of the factor was inhibited by mercurials, Cd²⁺, phenylarsine oxide, and diamide, indicating that it contains an essential dithiol. Dithiothreitol and dihydrolipoate did not replace the protein factor in stimulating the deficient particles. The purified dithiol-containing protein was precipitated and inhibited by antibody raised against coupling factor B. Since this antibody also inhibits coupling factor F₂, it is concluded that the active principle of coupling factors B and F₂ is the purified dithiol-containing protein of molecular weight 11–12 × 10³ referred to above.     

Electrical potential changes,H+ translocation and phosphorylation induced by short flash excitation in Rhodopseudomonas sphaeroides chromatophores

1. The basal decay of the carotenoid shift of chromatophores from photosynthetic bacteria following short flash excitation is approximately biphasic. The decay indicates the dissipation of the transmembrane electrical potential.2. The H⁺ efflux following rapid H⁺ binding after a flash, measured from the colour change of added cresol red, shows very similar kinetics to the carotenoid shift decay suggesting that the dissipation of the electric potential decay is a consequence of the H⁺ efflux.3. The electric potential decay is stimulated when the chromatophore suspension is supplemented with ADP and P_i (in either the presence or absence of antimycin A).4. The stimulated electric potential decay by ADP and P_i has a similar pH dependence to that of phosphorylation in continuous light.5. The stimulation of the electric potential decay by ADP and P_i is reversed, by aurovertin, an antibiotic which inhibits phosphorylation.6. The stimulation of the electric potential decay by ADP+P_i is also reversed by the inhibitors oligomycin and venturicidin. These inhibitors, but not aurovertin, also inhibit the fast phase of the decay under non-phosphorylating conditions.7. Valinomycin accelerates the overall rate of decay of the electric potential, inhibits the ADP and P_i stimulated electric potential decay, and inhibits the flash-induced phosphorylation. The decay rate of the H⁺ efflux however, is slower in the presence of this ionophore.8. Nigericin-type ionophores accelerate the overall decay rate of the H⁺ efflux and inhibit the ADP and P_i stimulated electric potential decay. The basal rate of the electric potential decay is unaffected by treatment with these ionophores.9. When a coupling factor associated with the chromatophore ATPase is removed from the membrane, both the stimulation of the electric potential decay by ADP and P_i and ADP phosphorylation are inhibited. Both reactions are completely restored after reconstitution with the crude coupling factor extract. The basal electric potential decay rate is not affected by the removal of coupling factor.     

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation,MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α₂ isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK_1/2 and PI3K/AKT; interaction between EGF receptor, α₁, and α₂ isoforms of Na,K-ATPase, Src, ERK_1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR⁸⁴⁵ and EGFR¹⁰⁶⁸; (ii) ammonia-induced ERK_1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK_1/2 phosphorylation; (iv) ammonia stimulates association between the α₁ Na,K-ATPase isoform, Src, EGF receptor, ERK_1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK_1/2, but in response to 3 mM ammonia it does not appear until after 12 h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.     

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10^?4 M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂‐induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂‐induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX‐2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley‐Liss, Inc.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y₂ receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y₂ receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca²⁺ mobilization indicating that nucleotides cause Ca²⁺ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca²⁺ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca²⁺ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca²⁺ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca²⁺ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca²⁺ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y₂ receptor can cause Ca²⁺ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.     

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca²⁺ mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀ ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca²⁺-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.     

Phosphorylation Regulates an Inwardly Rectifying ATP-sensitive K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>- Conductance in Proximal Tubule Cells of Frog Kidney

K⁺ channels in the renal proximal tubule play an important role in salt reabsorption. Cells of the frog proximal tubule demonstrate an inwardly rectifying, ATP-sensitive K⁺ conductance that is inhibited by Ba²⁺, G_Ba. In this paper we have investigated the importance of phosphorylation state on the activity of G_Ba in whole-cell patches. In the absence of ATP, G_Ba decreased over time; this fall in G_Ba involved phosphorylation, as rundown was inhibited by alkaline phosphatase and was accelerated by the phosphatase inhibitor F⁻(10 mM). Activation of PKC using the phorbol ester PMA accelerated rundown via a mechanism that was dependent on phosphorylation. In contrast, the inactive phorbol ester PDC slowed rundown. Inclusion of the PKC inhibitor PKC-ps in the pipette inhibited rundown. These data indicate that PKC-mediated phosphorylation promotes channel rundown. Rundown was prevented by the inclusion of PIP-2 in the pipette. PIP-2 also abrogated the PMA-mediated increase in rundown, suggesting that regulation of G_Ba by PIP-2 occurred downstream of PKC-mediated phosphorylation. G-protein activation inhibited G_Ba, with initial currents markedly reduced in the presence of GTPγs. These properties are consistent with G_Ba being a member of the ATP-sensitive K⁺ channel family.     

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

An acidic modification of the cytoplasmic domain contributes to the charge heterogeneity of the MHC class I antigens

 Polypeptide phosphorylation and sialylation of the glycan moieties contribute to the charge heterogeneity of the class I major histocompatibility complex glycoproteins. The present study demonstrates that a unique acidic modification unrelated to phosphorylation or glycosylation also affects the charge heterogeneity of the H2-K^k heavy chain of BW5147 lymphoma cells. In vitro cultivation of BW5147 cells results in changes in charge heterogeneity of the H2-K^k heavy chains due to the unique acidic modification. Sequential papain digestion of the 45 000 M _r H2-K^k glycoprotein yields a 42 500 M _r glycopolypeptide initially, followed by production of a 39 000 M _r glycopolypeptide. Results from experiments designed to localize and characterize the novel acidic modification suggest that the modification resides in the segment of the H2-K^k polypeptide located between the two papain cleavage sites. This portion of the polypeptide consists of the transmembrane region and part of the cytoplasmic domain of the H2-K^k heavy chain. At steady state, 25% of the total cell surface H2-K^k possesses this modification. In addition, the modification is mutually exclusive with the phosphorylation of the H2-K^k heavy chain at Ser-333. The possible biological significance of the novel modification of class I antigens is discussed. Received: 27 May 1997 / Revised: 10 September 1997     

Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP: MOLECULAR BASIS FOR ZN2+-INDUCED ERK MAPK ACTIVATION*

Excessive release of Zn²⁺ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn²⁺-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn²⁺ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn²⁺-mediated phosphorylation of STEP₆₁ at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP₆₁ at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP₆₁ toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn²⁺-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn²⁺-dependent neurotoxicity. The strong correlation between the temporal profile of STEP₆₁ hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP₆₁ through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn²⁺. The study provides further insight into the mechanisms of regulation of STEP₆₁ and also offers a molecular basis for the Zn²⁺-induced sustained activation of ERK2.     

Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc₃Man₉GlcNAc₂ (G₃M₉Gn₂) from the lipid-linked oligosaccharide (LLO) glucose₃mannose₉N-acetylglucosamine₂-P-P-dolichol (G₃M₉Gn₂-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2α (eIF2α) on Ser⁵¹ by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G₃M₉Gn₂-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2α Ser⁵¹ phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser⁵¹ kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.     

Modifications to histones immediately after synthesis

We have studied modifications occurring on histones immediately after synthesis. The labeling period was sufficiently brief such that the time interval between synthesis in the cytoplasm and deposition onto DNA can be easily detected. In this way it is possible to observe modifications occurring within seconds after arrival of the newly synthesized histone molecule in the nucleus. Histone F_2a1 is rapidly and extensively modified (in hepatoma tissue culture cells this is primarily by acetylation) so that the newly synthesized material appears primarily in the diacetyl modified form. The histone returns to a steady-state level of acetylation during the ensuing 30 minutes and this level is maintained throughout the lifetime of the cell. F₃ shows a somewhat similar pattern, except that it initially is less modified before it shifts to a steady-state level of modification. The lysinerich histone, F₁ is admitted to the nucleus with a modest level of phosphorylation. During the subsequent 60-minutes phosphorylation proceeds rapidly until the extent of phosphorylation typical of rapidly dividing cells is reached. This level of phosphorylation is then maintained in a steady-state against the action of a phosphatase. The rate of phosphorylation during the initial 60 minutes exceeds that at a later time by a factor of ~ ten. We argue that this observation accounts for previous observations of ³²P incorporation during G₁ and S phase and in the presence of inhibitors of DNA and histone synthesis.     

Requirements for different Ca2+ pools in the activation of rabbit platelets: I. Release reaction and protein phosphorylation

We examined the role of Ca²⁺, both extracellular and intracellular in origin, in the release reaction and protein phosphorylation in rabbit platelets stimulated with platelet activating factor (acetylglyceryl ether phosphorylcholine), thrombin, or ionophore A23187. In the presence of extracellular Ca²⁺, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ transport, blocked platelet activating factor-initiated serotonin release at a half-maximal inhibitor concentration of 40 μM, compared to 350 μM for thrombin-induced release and greater than 500 μM, for A23187-induced release. Platelet activating factor-induced phosphorylation of two platelet proteins of M_r=41 000 (P7P) and 20 000 (P9P) was inhibited by TMB-8, an effect which was additive to that caused by removing extracellular Ca²⁺. TMB-8 demonstrated only minor to non-existant inhibitory effects on phosphorylation in thrombin- or A23187-stimulated platelets. In contrast to P9P phosphorylation, phosphorylation of P7P caused by platelet activating factor was more dependent on a TMB-8 sensitive step than on the availability of extracellular Ca²⁺. Experiments with buffers containing fixed concentrations of free Ca²⁺ revealed that both processes (release and phosphorylation), when stimulated by platelet activating factor and thrombin, had the same threshold requirement (1–3 μM) for extracellular free Ca²⁺. These studies provide evidence that stimulation of rabbit platelets by platelet activating factor is more dependent on a TMB-8-sensitive intracellular Ca²⁺ source than is stimulation caused by thrombin. Furthermore, our data indicate that activation of different intracellular processes involved in platelet secretion (such as P7P and P9P phosphorylation) may require Ca²⁺ from different pools.     

The channel-kinase TRPM7 regulates phosphorylation of the translational factor eEF2 via eEF2-k

Protein translation is an essential but energetically expensive process, which is carefully regulated in accordance to the cellular nutritional and energy status. Eukaryotic elongation factor 2 (eEF2) is a central regulation point since it mediates ribosomal translocation and can be inhibited by phosphorylation at Thr56. TRPM7 is the unique fusion of an ion channel with a functional Ser/Thr-kinase. While TRPM7's channel function has been implicated in regulating vertebrate Mg²⁺ uptake required for cell growth, the function of its kinase domain remains unclear. Here, we show that under conditions where cell growth is limited by Mg²⁺ availability, TRPM7 via its kinase mediates enhanced Thr56 phosphorylation of eEF2. TRPM7-kinase does not appear to directly phosphorylate eEF2, but rather to influence the amount of eEF2's cognate kinase eEF2-k, involving its phosphorylation at Ser77. These findings suggest that TRPM7's structural duality ensures ideal positioning of its kinase in close proximity to channel-mediated Mg²⁺ uptake, allowing for the adjustment of protein translational rates to the availability of Mg²⁺.     

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca²⁺sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺([Ca²⁺]_i) due to plasmalemmal Ca²⁺influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca²⁺stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺]_iper se causes slowing of peptide chain elongation is lacking. L-type Ca²⁺channel activity of GH₃pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺]_ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺]_ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca²⁺channel antagonist, nisoldipine, lowered [Ca²⁺]_iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺]_ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca²⁺contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺]_ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca²⁺]_ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.