Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

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.     

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.     

A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase

A type 1 protein phosphatase from reticulocytes is shown to efficiently dephosphorylate the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase that phosphorylates the alpha subunit of eukaryotic peptide initiation factor 2, eIF-2. The kinase, activated in the presence of double-stranded RNA with concomitant phosphorylation of the Mr = 68,000 peptide, causes inhibition of peptide initiation and thereby effects translational control of protein synthesis. The Mn2+-dependent phosphatase is classified as a type 1 enzyme in that it is inhibited by inhibitor 2 in nanomolar concentrations and appears to have a Mr = 35,000 catalytic subunit. Dephosphorylation of the Mr = 68,000 peptide by the phosphatase is directly associated with a loss in kinase activity which can be restored by incubation with double-stranded RNA in the presence of ATP. The results demonstrate that the eIF-2 alpha kinase can undergo cyclic activation-inactivation that appears to be directly related to the phosphorylation state of the Mr = 68,000 peptide. They strongly support the previous conclusion that double-stranded RNA is required only for activation of the kinase and phosphorylation of the Mr = 68,000 peptide.     

The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein

Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.     

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Evidence for a second phosphorylation site on eIF-2 alpha from rabbit reticulocytes

Ser 51 in the NH2-terminal sequence of the alpha-subunit of eukaryotic peptide initiation factor 2 (eIF-2) has been identified as a second phosphorylation site for the heme-controlled eIF-2 alpha kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the alpha-subunit of spectrin. A synthetic peptide corresponding to eIF-2 alpha (41-54) is phosphorylated only in Ser 51 by the eIF-2 alpha kinase.     

Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase.

Translational initiation factor 3 (eIF-3) is phosphorylated by the cyclic AMP-regulated protein kinases from rabbit reticulocytes. eIF-3 is a large molecular weight complex which facilitates binding of the ternary complex containing met tRNA_f, GTP and initiation factor 2 to 40S ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40S ribosomal subunits and eIF-3, is controlled by cyclic AMP.     

Protease-activated kinase II as the potential mediator of insulin-stimulated phosphorylation of ribosomal protein S6

One of the earliest responses to insulin in target cells is stimulation of the phosphorylation of ribosomal protein S6. When exponentially growing 3T3-L1 cells are serum-starved, little phosphorylation of S6 is observed; however, following addition of insulin (10(-7) M), up to 5 phosphoryl groups are incorporated into S6. An enzyme mediating the insulin-stimulated phosphorylation of S6 has been identified as protease-activated kinase II. Two-dimensional peptide maps of tryptic digests of S6 from insulin-treated 3T3-L1 cells contain 5 phosphopeptides; the same 5 phosphopeptides are observed with tryptic digests of 40 S ribosomal subunits phosphorylated in vitro by protease-activated kinase II from rabbit reticulocytes. Protease-activated kinase II has also been identified and partially purified from the postribosomal supernatant of serum-starved and insulin-treated 3T3-L1 cells. The enzyme is present in the inactive proenzyme form in serum-starved cells; following insulin treatment, approximately 50% of the enzyme is in an activated form. Identical tryptic phosphopeptide maps are observed with these enzymes.     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Ca2+-independent activation of protease-activated kinase II by phospholipids/diolein and comparison with the Ca2+/phospholipid-dependent protein kinase

The proenzyme form of protease-activated kinase (PAK) II from reticulocytes has been shown to be activated in vitro by limited proteolysis and characterized using 40 S ribosomal subunits as substrate (T.H. Lubben and J.A. Traugh (1983) J. Biol. Chem. 258, 13992-13997). In these studies, we have shown that PAK II can be activated in a Ca2+-independent manner with phospholipids/diolein using histone 1, eukaryotic initiation factor 2, and 40 S ribosomal subunits as substrates. The addition of Ca2+ results in a diminution of PAK II activity. The Ca2+/phospholipid-dependent protein kinase (protein kinase C) is present in reticulocytes and is separated from PAK II during purification by chromatography on ADP-agarose. PAK II activated by limited proteolysis has the same substrate specificity as PAK II activated by phospholipids/diolein as shown by two-dimensional finger-printing of tryptic phosphopeptides of histone 1 and ribosomal protein S6, indicating proteolysis did not alter the specificity of the enzyme. Lipid vesicles decrease the Km of PAK II for histone 1 by 10-fold, while no effect is observed on the Km or the Vmax of PAK II for ATP. These results are strikingly different from the kinetics reported for protein kinase C, where the activators increase the Vmax for ATP. The two enzymes have similar, if not identical, substrate specificity with histone 1, as determined by phosphopeptide mapping, but at least 8-fold more protein kinase C than PAK II is required to incorporate a comparable amount of phosphate into S6 and it is not possible to incorporate stoichiometric amounts of phosphate into S6 with protein kinase C. The two protein kinases also differentially phosphorylate other substrates. The data support the hypothesis that PAK II and protein kinase C are closely related, but unique enzymes.     

Ribosomal S6 kinase p90rsk and mRNA cap-binding protein eIF4E phosphorylations correlate with MAP kinase activation during meiotic reinitiation of mouse oocytes

During meiotic reinitiation of the mouse oocyte, entry into M-phase is regulated by changes of protein phosphorylation and by the stimulation of selective mRNA translation following the nuclear membrane dissolution. Our results reveal that M-phase kinases (MAP kinase and histone H1 kinase) are being activated together with S6 kinase and with the phosphorylation of eIF4E, the cap-binding subunit of the initiation factor eIF-4F. In order to test which signaling pathway(s) is(are) involved, okadaic acid and cycloheximide have been used as tools for differentially modulating MAP and histone H1 kinase activities. A role for MAP kinases in the phosphorylation of eIF4E and the activation of S6 kinase is suggested. The possible implication of p90^rsk and/or of p70^s6k in the overall increase in S6 kinase activity has been examined. p70^s6k does not appear to be involved since phosphorylated forms are found in prophase and maturing oocytes. In contrast, p90^rsk is phosphorylated and activated in maturing oocytes. p90^rsk phosphorylation correlates with the activation of S6 kinase. These results suggest that the overall increase of S6 kinase activity is mostly due to p90^rsk activation. The roles of eIF4E phosphorylation and S6 kinase activation in the physiological induction of M-phase and in the okadaic acid-induced premature mitotic events are discussed. Mol. Reprod. Dev. 46:383–391, 1997. © 1997 Wiley-Liss, Inc.     

The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells

In eukaryotic cells, protein biosynthesis is controlled at the level of polypeptide chain initiation. During the initiation process, eukaryotic initiation factor 2 (eIF-2) catalyzes the binding of Met-tRNAf and GTP to the 40S ribosomal subunit. In a later step, eIF-2 is released from the ribosomal initiation complex, most likely as an eIF-2.GDP complex, and another initiation factor termed eIF-2B is necessary to recycle eIF-2 by displacing GDP by GTP. In rabbit reticulocytes, inhibition of protein synthesis is accompanied by the phosphorylation of the alpha-subunit of eIF-2, a process that does not render eIF-2 inactive, but prevents it from being recycled by eIF-2B. First described in rabbit reticulocytes as inhibitors of translation, two distinct eIF-2 alpha kinases are known: the haemin-controlled kinase (termed HCI) and the double-stranded RNA-activated kinase (termed DAI). eIF-2 alpha phosphorylation appears to be a reversible control mechanism since corresponding phosphatases have been described. Recent reports indicate a correlation between eIF-2 alpha phosphorylation and the inhibition of protein synthesis in several mammalian cell types under a range of physiological conditions. In this review, the physical and functional features of the known eIF-2 alpha kinases are described with respect to their role in mammalian cells and the mode of activation by cellular signals. Furthermore, the possible impact of the eIF-2/eIF-2B ratio and of the subcellular compartmentation of these factors (and the eIF-2 alpha kinases) on mammalian protein synthesis is discussed.     

The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase

The rabbit reticulocyte Mr 90,000 protein associated with the heme-sensitive eIF-2 alpha kinase has been identified previously as the mammalian heat shock protein of this size class (hsp 90). Purified reticulocyte hsp 90 when added exogenously to the kinase increases its activity. This stimulatory effect is abolished after incubation of hsp 90 with a highly purified type 1 phosphoprotein phosphatase isolated from reticulocytes. Phosphorylation of dephosphorylated hsp 90 by casein kinase II but not by cAMP-dependent protein kinase restores the biological activity of hsp 90 to stimulate eIF-2 alpha phosphorylation.     

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.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Isolation of a translational inhibitor from wheat germ with protein kinase activity that phosphorylates initiation factor eIF-2

A translational inhibitor (WGI) has been partially purified from wheat germ extracts. WGI inhibits protein synthesis in rabbit reticulocyte lysates with inhibition kinetics that are similar to those observed in heme-deficiency or by the addition of purified heme-regulated translational inhibitor (HRI). Initiation factor eIF-2 from rabbit reticulocytes overcomes this inhibition. This finding suggests that WGI inhibits protein chain initiation. WGI induced inhibition is enhanced by ATP (2 mM), and overcome by GTP (2 mM) and cyclic-AMP (10 mM). WGI preparations contain a cyclic-AMP independent protein kinase activity that phosphorylates the 38,000-dalton subunit of rabbit reticulocyte eIF-2. The phosphopeptide analyses of eIF-2 phosphorylated by WGI or HRI show that they phosphorylate the same site(s) of eIF-2. HRI phosphorylates the corresponding 38,000-dalton subunit of wheat germ eIF-2. These results obtained with WGI are similar to that of HRI. HRI has been identified as a cyclic-AMP independent protein kinase that phosphorylates the 38,000-dalton subunit of eIF-2 [for review see Ochoa, S. and de Haro, C. (1979) Ann. Rev. Biochem. $\text{48}$, 549]. Hence, these findings with wheat germ-a phylogenetically distant eukaryote, raise further the possibility that phosphorylation-dephosphorylation of eIF-2 may be an important general mechanism in the regulation of eukaryotic protein biosynthesis.     

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.     

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.