Mutation at the acidic residue-rich domain of eukaryotic initiation factor 2 (eIF2alpha)-associated glycoprotein p67 increases the protection of eIF2alpha phosphorylation during heat shock

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein p67 protects eIF2alpha phosphorylation from kinases. The N-terminal lysine-rich domains increase this activity and the acidic residue-rich domain inhibits it. Conserved amino acid residues D251, D262, E364, and E459 are involved in this inhibition. During heat shock, the overall protein synthesis rate decreases due to the increased levels of eIF2alpha phosphorylation. In this study, we examined whether the above inhibition is also found during heat shock. Indeed, the acidic residue-rich domain mutant (D6/2) showed a decreased level of eIF2alpha phosphorylation, and its second-site alanine substitutions at D251, D262, and E459 reversed this effect, whereas second-site alanine substitution at H331 and E364 residues further augmented it. A high-molecular-weight phosphoprotein and at least two faster-migrating phosphoproteins were detected by the monospecific polyclonal antibody against eIF2alpha(P) form in rat tumor hepatoma cells constitutively expressing the double mutant D6/2+D251A. Although the levels of p67 mutants were unaffected during heat shock, those of p67 and p67-deactivating enzyme varied. Furthermore, the overall rate of protein synthesis correlated with the level of eIF2alpha phosphorylation. Taken together, these results suggest that the lysine-rich domains and conserved amino acid residues of p67 are involved in the regulation of eIF2alpha phosphorylation during heat shock.     

The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2alpha from phosphorylation by kinases

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2alpha phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the D6/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2alpha and eIF2gamma and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.     

Protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.

The rate of protein synthesis in mammals is largely regulated by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2) that is modulated by the cellular glycoprotein, p67, due to its protection of eIF2alpha phosphorylation (POEP) activity. At the N-terminus of p67, there are three unique domains, and at the C-terminus there is a conserved amino acid sequence. To analyze the importance of these domains, C-terminal deletion mutants of rat p67 were expressed constitutively in KRC-7 cells. In these cells, the phosphorylation level of the alpha-subunit of eIF2 was determined, and it was found that expression of the 1-97 amino acid segment of rat p67 increases POEP activity in vivo, and induces the endogenous levels of p67. These cells also show increased growth rate, and efficient translation of chloramphenicol acetyltransferase and beta-galactosidase reporter genes. At the N-terminus of p67, there are two unique domains: a lysine-rich domain I with the sequence (36)KKKRRKKKK(44), and an acidic residue-rich domain with the sequence (77)EEKEKDDDDEDGDGD(91). Substitution of lysine-rich domain I with (36)NMKSGNKTQ(44) in rat recombinant p67 resulted in the inhibition of its POEP activity, and substitution of the acidic residue-rich domain with (77)QNIQKALEPEAGDGA(91), resulted in no inhibition of POEP activity in KRC-7 cells. Taken together, our data suggest that protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.     

Eukaryotic initiation factor 2-associated glycoprotein, p67, shows differential effects on the activity of certain kinases during serum-starved conditions

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. P67 has five conserved amino acid residues at the D251, D262, H331, E364, and E459 positions. To determine the roles of these conserved amino acid residues in eIF2alpha phosphorylation during serum-starved conditions, we constitutively expressed D251A, D262A, H331A, E364A, and E459A mutants in rat tumor hepatoma cells. We find that the point mutants D251A, H331A, and E364A lower the levels of eIF2alpha phosphorylation. These low levels of phosphorylation decrease when serum-starved cells are grown in medium containing serum. To understand the mechanism of action of the p67 mutants in eIF2alpha phosphorylation during serum-starvation, we performed detailed biochemical analyses with the D251A mutant. We find that neither the O-GlcNAc modification on the D251A mutant nor the binding of D251A mutant with eIF2gamma has significant effects on eIF2alpha phosphorylation during serum-starved conditions. However, the D251A mutant inhibits p67's activity to suppress the activity of ERK1/2. Our data suggest that both p67 and the D251A mutant bind to ERK1, thus strengthening the idea that p67 regulates the activity of ERK1. During serum-starvation conditions, both PKR and PERK are phosphorylated and the D251A mutant shows increased stability of PERK as well as a slight decrease in its activity. Altogether, our data provide evidence to suggest that p67 modulates the expression and activity of certain eIF2alpha-specific kinases.     

MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms

Methionine aminopeptidases (MAPs) play important roles in protein processing. MAPs from various organisms, for example E. coli, S. typhimurium, P. furiosus, Saccharomyces cerevisiae, and porcine have been purified to homogeneity and their MAP activities have been tested in vitro and in vivo. The DNA sequence analyses of MAP genes from the above organisms reveal sequence homologies with other prokaryotic MAPs as well as with various eukaryotic homologues of rat p67. The cellular glycoprotein, p67 protects the alpha-subunit of eukaryotic initiation factor 2 (eIF2) from phosphorylation by its kinases. We call this POEP (protection of eIF2alpha phosphorylation) activity of p67. The POEP activity of p67 is observed in different stress-related situations such as during heme-deficiency of reticulocytes, serum starvation and heat-shock of mammalian cells, vaccinia virus infection of mammalian cells, baculovirus infection of insect cells, mitosis, apoptosis, and possibly during normal cell growth. The POEP activity of p67 is regulated by an enzyme, called p67-deglycosylase (p67-DG). When active, p67-DG inactivates p67 by removing its carbohydrate moieties. Remarkable amino acid sequence similarities at the C-terminus of rat p67 with its eukaryotic and prokaryotic homologues which have MAP activities, raise several important questions: i) does rat p67 have MAP activity?; and ii) if it does have MAP activity, how the two activities (POEP and MAP) of p67 are used by mammalian cells during their growth and differentiation. In this review, discussions have been made to evaluate both POEP and MAP activities of p67 and their possible involvement during normal growth and cancerous growth of mammalian cells.     

Autoproteolysis of rat p67 generates several peptide fragments: the N-terminal fragment, p26, is required for the protection of eIF2alpha from phosphorylation

Eukaryotic initiation factor 2-associated glycoprotein, p67, plays important roles in the regulation of eIF2alpha phosphorylation and thus maintains cell growth and proliferation. The p67 sequence can be divided into two segments, the N-terminal segment of amino acids 1-107 (p26) and the downstream segment of amino acids 108-480 (p52). Comparison of the amino acid sequences of p67 from lower to higher organisms suggests that there is a progressive addition of several unique domains at the N-terminus of p67, and these unique domains, which are present in p26, play important roles in the modulation of eIF2alpha phosphorylation in mammalian cells. To test the hypothesis that the p26 segment is generated from p67 due to its autoproteolysis and whether p26 is required for the protection of eIF2alpha from phosphorylation, we have analyzed the time-dependent cleavage of functionally active rat recombinant p67 purified from either baculovirus-infected insect cells or transiently transfected mammalian cells. We noticed a regulated cleavage of p67 that generates several peptides along with the most stable p26 and p52 fragments. The p52 fragment has a low level of autoproteolysis activity that possibly increases the autoproteolysis of full-length p67. This activity could not be inhibited by a serine protease inhibitor, PMSF, but could be inhibited by a cocktail of protease inhibitors that includes bestatin, leupeptin, E64, AEBSF, and aprotinin. To provide evidence that the fragmentation of p67 is not due to the presence of any contaminant protease(s), we fractionated purified rat p67 with molecular sieve, anion exchange, and cation exchange chromatographic steps performed in the presence of different K+ ion concentrations. Our data show that the extent of cleavage of p67 into different fragments is higher in the presence of 0.75 M K+ ion and in samples stored at -80 degrees C. Under parallel conditions, p67's mutants, D251A and D262A, exhibited very little to no cleavage, whereas the H231E mutant exhibited extensive cleavage that generated a large amount of p26 fragment. The p26 fragment exhibited protection of eIF2alpha phosphorylation both in vivo and in vitro. Altogether, our data provide evidence that rat p67 has autoproteolytic activity that generates p26, which is required to block eIF2alpha from phosphorylation.     

Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.     

The N-terminal lysine residue-rich domain II and the 340-430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the gamma-subunit of eIF2

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, plays an important role in protecting eIF2alpha from phosphorylation by eIF2alpha-specific kinases. To understand the molecular details of interaction between p67 and the subunits of eIF2, we applied several biochemical and mutational analyses to identify interacting domains within p67 and eIF2gamma. These studies were combined with functional in vivo and in vitro assays to address the importance of the interactions between p67 and eIF2gamma in eIF2alpha phosphorylation. Studies from yeast two-hybrid assays show that p67 interacts strongly with eIF2gamma, relatively weakly with eIF2alpha, and no interaction with eIF2beta. Further mutational analyses provided evidence that the N-terminal lysine-rich domain II and the 340-430 amino acid segment of p67 interact strongly with the C-terminal 409-472 amino acid segment of eIF2gamma. GST pull-down assays show that the interaction between p67 and eIF2gamma is direct. From co-immunoprecipitation studies, we find that the interaction between p67 and eIF2gamma could not only be detected in mammalian cells growing in growth medium, it could also be detected in transiently transfected cells with expression plasmids encoding p67 and eIF2gamma. However, this interaction could not be detected in p67 mutants lacking lysine-rich domain II and the 340-430 amino acid segment. We also find a very good correlation between p67 binding to eIF2gamma and the protection of eIF2alpha from phosphorylation. Altogether, our data provide genetic evidence for the interaction between p67 and eIF2gamma and that this interaction modulates the phosphorylation of eIF2alpha.     

Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.     

Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.     

Investigation of conserved acidic residues in 3-hydroxy-3-methylglutaryl-CoA lyase: implications for human disease and for functional roles in a family of related proteins

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metal-dependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280. HMG-CoA lyase deficiency results from a human mutation that substitutes lysine for glutamate 279. The E279K mutation has also been engineered; expression in Escherichia coli produces an unstable protein. Substitution of alanine for glutamate 279 produces a protein that is sufficiently stable for isolation and retains substantial catalytic activity. However, thermal inactivation experiments demonstrate that E279A is much less stable than wild-type enzyme. HMG-CoA lyase deficiency also results from mutations at aspartate 42. Substitutions that eliminate a carboxyl group at residue 42 perturb cation binding and substantially lower catalytic efficiency (104-105-fold decreases in specific activity for D42A, D42G, or D42H versus wild-type). Substitutions of alanine for the other conserved acidic residues indicate the importance of glutamate 72. E72A exhibits a 200-fold decrease in kcat and >103-fold decrease in kcat/Km. E72A is also characterized by inflation in the Km for activator cation (26-fold for Mg2+; >200-fold for Mn2+). Similar, but less pronounced, effects are measured for the D204A mutant. E72A and D204A mutant proteins both bind stoichiometric amounts of Mn2+, but D204A exhibits only a 2-fold inflation in KD for Mn2+, whereas E72A exhibits a 12-fold inflation in KD (23 microm) in comparison with wild-type enzyme (KD = 1.9 microm). Acidic residues corresponding to HMG-CoA lyase Asp-42 and Glu-72 are conserved in the HMG-CoA lyase protein family, which includes proteins that utilize acetyl-CoA in aldol condensations. These related reactions may require an activator cation that binds to the corresponding acidic residues in this protein family.     

Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site.     

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.     

Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E‐φ‐G‐D‐[KR]‐[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca²⁺ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site‐directed mutagenesis, we generated 17 mutations in the yeast Golgi‐localized Ca²⁺ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca²⁺‐ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.     

Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus.

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.     

Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein

AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining, ³⁵S-Met/Cys metabolic labeling, Western blotting analysis, sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells. Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR). Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins. In vivo ³²P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation, eIF2α phosphorylation, and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus. In a sucrose gradient, approximately 30% of the overexpressed p67 was bound with ribosomes. p67 interacted with the kinase domain, but not the dsRNA-binding domains of PKR. Only the glycosylated p67 was associated with the ribosome, and p67 did not compete with PKR for ribosome binding. In breast cancer cells, there was increased autophosphorylation of PKR but no phosphorylation of eIF2α, compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.     

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.     

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173).     

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.