Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.     

Phosphorylation of elongation factor 1 beta by an endogenous kinase affects its catalytic nucleotide exchange activity

Elongation factor 1 beta (EF-1 beta) from Artemia is phosphorylated to a high percentage at serine 89 by an endogenous kinase present in EF-1 beta gamma. Protein sequencing of EF-1 beta revealed that this serine residue is located N-terminally of an acidic cluster of amino acids, (formula; see text) which is critical for casein kinase II-type substrate recognition. A number of compounds known to influence casein kinases were studied, revealing that the kinase activity as present in EF-1 beta gamma belongs to the class of casein kinase II. The rate of nucleotide exchange on EF-1 alpha as catalyzed by EF-1 beta was found to be affected reversibly by the state of phosphorylation of EF-1 beta. In the presence of dephosphorylated EF-1 beta, the exchange rate is almost twice as large compared to the rate in the presence of phosphorylated EF-1 beta. Rephosphorylation of dephosphorylated EF-1 beta diminishes the activity of the protein again. The role of casein kinase II-type enzymes in modulating the function of proteins involved in polypeptide synthesis is discussed.     

Trypanosoma cruzi: in vitro phosphorylation of tubulin by a protein kinase CK2-like enzyme

One predominant 55-kDa polypeptide was phosphorylated in vitro in Trypanosoma cruzi homogenates prepared from three differentiation stages: epimastigotes, trypomastigotes, and spheromastigotes. Anti-alpha and anti-beta tubulin monoclonal antibodies immunoprecipitated the phosphorylated 55-kDa polypeptide from epimastigote extracts. Phosphoserine was the only residue phosphorylated in vitro in the 55-kDa polypeptide and in immunoprecipitated alpha tubulin. The phosphorylation of both the 55-kDa polypeptide and exogenously added casein was inhibited with GTP, heparin, and 2,3-bisphosphoglycerate in a dose-dependent manner, indicating the involvement of a CK2-like protein kinase. Moreover, when tubulin was isolated from an epimastigote homogenate by ultracentrifugation, followed by DEAE-Sephacel chromatography, a protein kinase that phosphorylated tubulin and casein co-purified with this cytoskeletal component. This result suggests an association between tubulin and its corresponding protein kinase in T. cruzi.     

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.     

Red cells of newborn rats have low bisphosphoglyceromutase and high pyruvate kinase activities in association with low 2,3-bisphosphoglycerate

2,3-Bisphosphoglycerate is a physiologically important regulator of red cell oxygen affinity during mammalian development. The rat has no fetal hemoglobin, but the newborn red cell has low 2,3-bisphosphoglycerate and high ATP concentrations, and high oxygen affinity. This report shows that red cell bisphosphoglyceromutase activity increases from near zero in the newborn rat to very high levels by four weeks of age. This increase roughly parallels the increase in red cell 2,3-bisphosphoglycerate concentration. Red cell pyruvate kinase activity declines ten-fold from birth to four weeks of age. This decrease is associated with a changeover in red cell populations from larger to smaller cells. The glycolytic rate is at least 50% higher in newborn than adult rat red cells. The data suggest that high pyruvate kinase activity and glycolytic rate contribute to the high ATP concentration in newborn rat red cells, but that their low 2,3-bisphosphoglycerate concentration is due primarily to low bisphosphoglyceromutase activity.     

B23 is a downstream target of polyamine- modulated CK2

Our previous studies have shown that the overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases the enzymatic activity of the polyamine-responsive enzyme casein kinase 2 (CK2). Because CK2 is known to preferentially associate with the nuclear matrix in response to other trophic stimuli, we investigated the effects of ODC overexpression on CK2 localisation and on the CK2-mediated phosphorylation of a known CK2 substrate, the nucleolar phosphoprotein B23. Immunofluorescence analysis of CK2 and B23 in primary keratinocytes revealed that ODC overexpression resulted in the colocalisation of CK2 with B23 at the nucleolar borders. ODC overexpression also increased CK2 kinase activity 2-fold at the nuclear matrix, a response which could be abrogated by treatment of K6/ODC transgenic keratinocytes with the ODC inhibitor α-difluoromethylornithine (DFMO). Levels of B23 protein were also elevated in ODC-overexpressing cells compared to normal cells or transgenic cells treated with DFMO. This increase in protein level was neither due to an increase in steady-state mRNA levels, nor was it due to increased stability of B23 protein. Phosphorylation of B23 was also increased in ODC-overexpressing cells, and this increased phosphorylation could be blocked by treatment of the cells with the CK2 kinase inhibitors apigenin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). These data suggest that B23 may be a downstream effector of polyamines via phosphorylation by the protein kinase CK2.     

Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.     

Epidermal growth factor stimulates protein kinase activity, specific for ribosomal protein S6 in loach embryo blastoderm

The activity of a protein kinase specific to ribosomal protein S6 was determined in early loach embryos in basal conditions and after their treatment with epidermal growth factor (EGF). The cytosol of loach blastoderms isolated at the early gastrula stage possessed a high level of protein kinase activity catalysing incorporation of 0. 33 pmol.min-1.mg-1 of Pi into exogenous S6 protein of rat liver ribosomal 40S subunit. The treatment of embryos for 30 min with EGF (10 ng/ml) added to the incubation medium caused an 25% increase of total S6-kinase activity in cytosol compared with its counterpart in non-stimulated embryos. After chromatography of loach embryos cytosol on DE-52 three fractions possessing S6-kinase activity were revealed, which were eluted with 10 microM cAMP (I), 150 mM NaCl (II) and 300 mM NaCl (III), respectively. After treatment of blastoderms with EGF in the described conditions the enzymatic activity 2-fold decreased in fraction I, increased in fraction II 4-fold and remained practically unchanged in fraction III. The mitogen-stimulated kinase, apart from S6 protein, phosphorylated also casein and but not histone H1.     

Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization

Mammalian casein kinase 1delta (CK1delta) is a homologue of the S. cerevisiae Hrr25p protein kinase. Hrr25p is involved in regulating diverse events including vesicular trafficking, gene expression, DNA repair, and chromosome segregation. In contrast to Hrr25p, little is known about the function, regulation, or subcellular localization of CK1delta. In the present study, we show that CK1delta in mammalian cells is mainly cytoplasmic and enriched within the Golgi and/or ER-Golgi transport vesicles, consistent with a role in vesicular trafficking. Transient expression of green fluorescent protein (GFP)- or FLAG peptide-tagged CK1delta showed localization similar to that of the endogenous CK1delta. GFP-CK1delta was also enriched at the centrosomes in interphase cells. Strikingly, two inactive mutant CK1delta proteins (K38M and T176I) showed almost exclusive nuclear staining, suggesting that protein kinase activity is required for normal localization of CK1delta and prevention of nuclear accumulation. The nuclear export inhibitor leptomycin B promoted nuclear enrichment of CK1delta indicating that nuclear localization of CK1delta occurs physiologically. Both endogenous CK1delta and GFP-CK1delta are enriched on the spindle poles in mitotic cells, consistent with a role in regulating spindle formation. Localization is a property of the protein kinase domain and is independent of the C-terminal noncatalytic domain. These data are consistent with roles for CK1delta in mammalian cells analogous to those of its yeast counterparts.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

Studies on avian erythrocyte metabolism. XVI. Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes

The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined. Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate. Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h. Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension. Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions. The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation). Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions. The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr. The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)

CK1delta, a member of the casein kinase 1 family of serine/threonine specific kinases, has been shown to be involved in the regulation of microtubule dynamics. We have now identified a 176 aa fragment of the light chain LC2 of MAP1A (termed LC2-P16) specifically interacting with CK1delta. Two CK1delta interacting domains of LC2 were identified, located between aa 2629 and 2753 close to aa 2683 and between aa 2712 and 2805 of LC2. The two regions necessary for the interaction of LC2 with CK1delta have been mapped between aa 76-103 and aa 351-375 of CK1delta. Furthermore, LC2 has been identified as a new substrate of CK1delta. We therefore propose a model in which CK1delta could modulate microtubule dynamics by changing the phosphorylation status of the light chain LC2 of MAP1A.     

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.     

Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1

In meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation, called meiosis I and II. Disjunction of maternal from paternal centromeres during meiosis I depends on the attachment of sister kinetochores to microtubules emanating from the same pole. In budding yeast, monopolar attachment requires recruitment to kinetochores of the monopolin complex. How monopolin promotes monopolar attachment was unclear, as its subunits are poorly conserved and lack similarities to proteins with known functions. We show here that the monopolin subunit Mam1 binds tightly to Hrr25, a highly conserved casein kinase 1 delta/varepsilon (CK1delta/varepsilon), and recruits it to meiosis I centromeres. Hrr25 kinase activity and Mam1 binding are both essential for monopolar attachment. Since CK1delta/varepsilon activity is important for accurate chromosome segregation during meiosis I also in fission yeast, phosphorylation of kinetochore proteins by CK1delta/varepsilon might be an evolutionary conserved process required for monopolar attachment.     

Tyrphostin AG213对重组人蛋白激酶CK2全酶的抑制动力学

利用基因工程克隆、表达和纯化获得重组人蛋白激酶CK2α和 β亚基 ,在体外等摩尔数混合构成有最大生物活性的重组人CK2全酶 .以重组人CK2全酶为分子靶点 ,研究tyrphostinAG2 13对该全酶的直接作用及其抑制动力学 .通过测定转移到CK2底物上的 [γ 3 2 P]GTP的 [3 2 P]放射活度 ,检测CK2活性 .结果表明 :重组人CK2是一种Ca2 + 、cAMP和cGMP等第二信使非依赖性蛋白激酶 ,与天然CK2的性质一致 .AG2 13对重组人CK2全酶具有很强的抑制作用 ,IC50 为 1 1μmol L ,抑制作用远大于已知CK2的抑制剂 5 ,6 二氯 1 β 呋喃糖苯并咪唑 (DRB)和N (2 氨乙基 ) 5 氯萘 1 硫胺 (A3) .AG2 13对重组人CK2全酶的动力学研究表明 :它与GTP呈现非竞争 竞争性混合型抑制作用 ,抑制常数Ki 和Ki′值分别为 0 6 μmol L与 1 4 μmol L ;与酪蛋白呈非竞争性抑制作用 ,Ki 值为 0 9μmol L .结果说明 ,tyrphostinAG2 13不仅是酪氨酸蛋白激酶的抑制剂 ,而且是一种十分有效的蛋白激酶CK2的抑制剂 .重组人蛋白激酶CK2可作为一种较为简便筛选和开发有效的CK2抑制剂的分子靶点 .     

Mayaro virus infection cycle relies on casein kinase 2 activity

Replication of Mayaro virus in Vero cells induces dramatic cytopathic effects and cell death. In this study, we have evaluated the role of casein kinase 2 (CK2) during Mayaro virus infection cycle. We found that CK2 was activated during the initial stages of infection ( approximately 36% after 4h). This activation was further confirmed when the enzyme was partially purified from the cellular lysate either by Mono Q 5/5Hr column or heparin-agarose column. Using this later column, we found that the elution profile of CK2 activity from infected cells was different from that obtained for control cell enzyme, suggesting a structural modification of CK2 after infection. Treatment of infected cells with a cell-permeable inhibitor of CK2, dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), abolished the cytopathic effect in a dose-dependent manner. Together this set of data demonstrates for the first time that CK2 activity in host cells is required in Mayaro virus infection cycle.     

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.