Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C

Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.     

Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F

Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.     

Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap

Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.     

Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, was isolated by m7 GTP-Sepharose affinity chromatography from rabbit reticulocytes incubated with [32P]orthophosphate. Following treatment of reticulocytes with phorbol 12-myristate 13-acetate (PMA) for 30 min, stimulation of phosphorylation of both the p25 and p220 subunits was observed (2.5-5-fold). Two variants were observed for p25 in the absence and presence of PMA when analyzed by two-dimensional gel electrophoresis. Only the more acidic of these was phosphorylated, with the level of phosphorylation increased upon PMA treatment. One main variant was observed for p220; following PMA stimulation, in addition to increased labeling of this variant, two more acidic phosphorylated variants were observed. Low levels of eIF-3 and -4B were associated with purified eIF-4F, and PMA treatment stimulated phosphorylation of eIF-3 (p170) by 2-4-fold and eIF-4B by 1.5-2.5 fold. Two-dimensional phosphopeptide mapping of p25 phosphorylated in the absence or presence of PMA generated a single tryptic phosphopeptide, suggesting a single phosphorylation site. A more complex phosphopeptide map was observed with p220 subunit. The maps for both subunits contained the same phosphopeptides as those obtained when eIF-4F was phosphorylated in vitro by the Ca2+/phospholipid-dependent protein kinase, indicating this protein kinase directly modulated eIF-4F in response to PMA.     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.     

Identification of an isozyme form of protein synthesis initiation factor 4F in plants.

We showed previously that wheat germ extracts contain two forms of protein synthesis initiation factor 4F that have very similar functional properties (Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232). One form, designated eIF-4F, is a complex containing two subunits, p220 and p26. The other form, designated eIF-(iso)4F, is a complex containing two subunits, p82 and p28, which are antigenically distinct from the subunits of eIF-4F. Both the p26 subunit of eIF-4F and the p28 subunit of eIF-(iso)4F are m7G cap-binding proteins. In this investigation, affinity-purified antibodies to the p220 and p26 subunits of wheat germ eIF-4F and to the p82 and p28 subunits of wheat germ eIF-(iso)4F were used to determine if isozyme forms of eIF-4F are present in maize and cauliflower. Extracts from wheat germ, maize root tips, and cauliflower inflorescences were partially purified by adsorption on m7GTP-Sepharose and elution with m7GTP (MGS eluate). Analysis by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies to the subunits of the wheat germ factors showed that the MGS eluate from maize contains polypeptides that react with antibodies to the p82 and p28 subunits of wheat eIF-(iso)4F, as well as polypeptides that react with antibodies to the p220 and p26 subunits of wheat eIF-4F. The MGS eluate from cauliflower also contains polypeptides that reacted with antibodies to the subunits of wheat eIF-(iso)4F. These results indicate that both maize and cauliflower contain the isozyme form of eIF-4F. In addition, it was found that the factors in the MGS eluate from maize support polypeptide synthesis in a system from wheat deficient in eIF-4F and eIF-(iso)4F, whereas the factors in the MGS eluate from cauliflower support polypeptide synthesis only to a small extent.     

Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.     

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.     

Comparative analysis of phosphorylation of translational initiation and elongation factors by seven protein kinases

Four initiation factors (eIF-2, -3, -4B, and -4F), previously shown to be phosphorylated in vivo, are each phosphorylated to a significant extent in vitro (greater than 0.3 mol of phosphate/mol of factor) by at least three different protein kinases. An S6 kinase from liver, an active form of protease-activated kinase II which modifies the same sites on S6 as those phosphorylated in vivo in response to mitogens, phosphorylates the beta subunit of eIF-2, eIF-3 (p120-p130), eIF-4B, and eIF-4F (p220). The Ca2+, phospholipid-dependent protein kinase phosphorylates eIF-2 beta, eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220, p25). The cAMP-dependent protein kinase significantly modifies eIF-4B and, to a lesser extent, eIF-3 (p130). Casein kinase I incorporates phosphate only into eIF-4B, but to a limited extent. Casein kinase II phosphorylates eIF-2 beta, eIF-3 (p170, p120), and eIF-4B, while protease-activated kinase I modifies eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220). The mitogen-stimulated S6 kinase from 3T3-L1 cells, activated in response to insulin, does not phosphorylate any of the initiation factors. There is no significant incorporation of phosphate into eIF-2 alpha or -gamma, eIF-4A, eIF-4C, eIF-4D, EF-1, or EF-2 by any of the protein kinases examined. Phosphopeptide mapping of tryptic digests of the phosphorylated subunits shows that the individual protein kinases modify different sites. The sites phosphorylated in vitro reflect those modified in vivo as shown with eIF-4F in concomitant studies with reticulocytes treated with tumor-promoting phorbol ester (Morley, S.J., and Traugh, J. A. J. Biol. Chem., in press). Thus, we have identified multipotential protein kinases which modify four initiation factors phosphorylated in vivo and have shown that phosphorylation of these translational components can be coordinately regulated.     

Variations in cap-binding complexes from uninfected and poliovirus-infected HeLa cells

Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.     

Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Purification and characterization of a 400-kDa nonhistone chromatin protein that serves as an effective phosphate acceptor for casein kinase II from Ehrlich ascites tumor cells

A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.     

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.     

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity

Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.     

The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase

In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.     

Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein

The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.     

Separation of protein synthesis initiation factor eIF4A from a p220-associated cap binding complex activity

A cap binding complex activity was purified from HeLa cells by a procedure which does not depend on the use of cap-affinity chromatography. The activity co-purified with a Mr 220000 polypeptide (p220), but not with elF4A. The active complex therefore differs from eIF4F, the complex purified by cap analog-affinity chromatography, in that it lacks the Mr 50000 subunit which is antigenically identical to elF4A. The activities of elF4F, CBP I and the eIF4A free complex purified here were compared in a fractionated system translating capped globin mRNA. Results indicate that the two complexes have similar activities and that they perform a function which cannot be provided by CBP I alone. Cap binding complex activity can be partly separated from eIF4A activity on sucrose gradients, thus eIF4A provides a function that is distinct from cap binding complex activity. The results indicate that eIF4A can be physically separated from the cap binding complex without affecting the ability of the remaining structure to function in an in vitro translation system. They suggest that the eIF4A-free complex may provide a function that is not a property of either CBP I or of eIF4A, but may be a property of p220.     

A mouse homolog to the avian sarcoma virus src protein is a member of a protein kinase cascade

Recent work has identified a cascade of membrane bound protein kinases in Ehrlich ascites tumor cells. These enzymes, designated PKL, PKS and PKM, are present in both Ehrlich tumor and mouse brain, but the cascade is active only in the tumor tissue. We have now purified a fourth protein kinase, PKF, that is also associated with this cascade. Protein kinase F prosphorylates PKL and is phosphorylated by PKS. The position of this kinase in the cascade is as follows, where the arrows denote phosphorylation: [Formula: see text] The phosphorylation by PKF, like phosphorylation by the other kinases, is at a tyrosine residue and causes the substrate kinase (PKL) to become active. The role of the tyrosine phosphorylation in activating these kinases is described in detail elsewhere. One result of activation of the cascade is the phosphorylation of the beta subunit of the Na+K+-ATPase, which causes inefficient Na+ pumping and is at last in part responsible for the high aerobic glycolysis of Ehrlich ascites tumor cells. By several criteria protein kinase F from Ehrlich cells is homologous to the src gene product (pp60src) from avian sarcoma viruses. Antiserum raised against PKF and sera from rabbits bearing rous sarcoma virus (RSV)-induced tumors quantitatively precipitate the same 60 kd phosphoprotein from cell lysates of three different RSV-transformed cell lines. Both proteins phosphorylate PKL and a 130 kd cytoskeletal protein (vinculin). The tryptic maps of these proteins are closely similar. Both proteins bind specifically to PKL covalently coupled to Sepharose. We used this latter observation to facilitate the purification of pp60 src from RSV-transformed cells.