Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.     

Presence of queuine in Drosophila melanogaster: correlation of free pool with queuosine content of tRNA and effect of mutations in pteridine metabolism.

Queuine, a modified form of 7-deazaguanine present in certain transfer RNAs, is shown to occur in Drosophila melanogaster adults in a free form and its concentration varies as a function of age, nutrition and genotype. In several, but not all mutant strains, the concentrations of queuine and the Q(+) (queuine-containing) form of tRNATyr are correlated. The bioassay employs L-M cells which respond to the presence of queuine by an increase in their Q(+)tRNAAsp that is accompanied by a decrease in the Q(-)tRNAAsp isoacceptors. The increase in Q(+)tRNATyr in Drosophila that occurs on a yeast diet is accompanied by an increase in queuine. Similarly the increase of Q(+)tRNAs with age also is accompanied by an increase in free queuine. In two mutants, brown and sepia, these correlations were either diminished or failed to occur. Indeed, the extract of both mutants inhibited the response of the L-M cells to authentic queuine. When the pteridines that occur at abnormally high levels in sepia were used at 1 x 10(-6)M, the inhibition of the L-M cell assay occurred in the order biopterin greater than pterin greater than sepiapterin. These pteridines were also inhibitory for the purified guanine:tRNA transglycosylase from rabbit but the relative effectiveness then was pterin greater than biopterin greater than sepiapterin. Pterin was competitive with guanine in the enzyme reaction with Ki = 0.9 x 10(-7)M. Also when an extract of sepia was chromatographed on Sephadex G-50, the pteridine-containing fractions only were inhibitory toward the L-M cell assay or the enzyme assay. These results indicate that free queuine occurs in Drosophila but also that certain pteridines may interfere with the incorporation of queuine into RNA.     

Rapid expression of novel proteins in goldfish retina following optic nerve crush

Polyadenylated messenger RNA was isolated from goldfish retinas at various times following unilateral crush of the optic nerve. RNA was translated in a cell-free system and product proteins analyzed by two-dimensional gel electrophoresis and autofluorography. Poly(A)+ mRNA-directed protein synthesis revealed an 8-fold increase in the labeling of polypeptides of about 30 kd Mr and a pI of 5.5 in retinas 2 d following optic nerve crush, compared with control retina mRNA translation products. In vitro labeling of retinal proteins revealed the enhanced synthesis of comparable 30 kd proteins in 2 d post-crush retinas. Evidence presented suggests that this 30 kd protein cluster may correspond to fish 30 kd stress or heat-shock proteins (hsp-30).     

Formation of queuine-containing tRNATyr in higher plants

Lupin and parsley cells cultured in vitro in defined media are not able to synthesize queuine-containing tRNA^Tyr. Addition of free queuine to their culture media has brought about complete (80–100%) or high (45–58%) conversion of (Q−)-into (Q+)tRNA^Tyr in lupin and parsley cultures, respectively.(Q+)tRNA^Tyr does not occur in young leaves and stems of lupin albeit it does in its seeds. It may suggest, although indirectly, that (Q+)tRNA^Tyr could play some function during embryo development, but probably not in other plant organs.We suggest that the formation of (Q+)tRNAs in higher plants depends on the delivery of queuine of bacterial origin from the environment.     

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.     

Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii.

Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.     

Modulation of Lactate Dehydrogenase Isozymes by Modified Base Queuine

The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA_GUN) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.     

Effect of estrogen-treated porcine ampulla oviductal epithelial cells on early embryonic development in vitro and characterization of their protein synthetic activity

Recent studies by Buhi et al. have demonstrated that estrogen (E₂) is responsible for the induction of de novo synthesis and secretion of certain oviductal secretory proteins (OSP) and inhibition of other OSP in porcine oviductal explant cultures. The present work was undertaken to evaluate the effect of E₂-treated oviductal epithelial cell coculture on the development of early porcine embryos derived from in vitro matured and fertilized oocytes. In vitro synthesis of secretory proteins by E₂-treated oviductal cells used for coculture was also investigated by one-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The results showed that the cleavage rate was significantly enhanced by coculturing fertilized eggs with E₂-treated oviductal epithelial cells. The in vitro protein synthetic pattern of oviductal secretory proteins was influenced by E₂ treatment. These variations included the disappearance of one protein (82 000 M_r) and the appearance of another (33 000 M_r) in the E₂-treated group as assessed by 1D-SDS-PAGE. Additional proteins of M_r 97 000 and an M_r 36 000–45 000 complex were increased in abundance by the E₂ treatment. Analyses by 2D-SDS-PAGE revealed three major E₂-dependent proteins, of M_r 45 000 (pI 5.5), 43 000 (pI 5.5) and a 36 000–45 000 M_r (pI 4.8) protein complex, whereas polypeptides of M_r 97 000 (pI 5.1), 36 000 (pI 8.0) and 25 000 (pI 6.8) were inhibited by E₂ treatment. The results demonstrated that porcine epithelial cell protein synthetic patterns are influenced by E₂ treatment and that estradiol treatment of oviductal cells may increase the rate of zygote cleavage during early development in vitro in pigs.     

Proteins associated with B lymphocyte hyperactivity in New Zealand black mice

New Zealand Black (NZB) mice exhibit polyclonal B cell activation and elevated immunoglobulin production, an abnormality associated with the spontaneous autoimmune disease that affects this strain. To further our understanding of this abnormality of B cell differentiation and maturation, we have employed two-dimensional polyacrylamide gel electrophoresis to analyze the proteins synthesized by lymphocytes of several strains. Two proteins were produced by lymphocytes from NZB mice but not those from normal strains. One was a 16 kd protein with a pI of 5.1, and the other was a 27.5 kd protein with a pI of 4.5. The presence of the xid gene on the NZB background suppressed production of both proteins. They were synthesized by spleen cells but not by bone marrow or lymph node cells, and production was restricted to enlarged B lymphocytes. p16 was synthesized by normal mouse strain B cells upon stimulation with LPS. The 27.5 kd protein was shown to be secreted. On the basis of partial amino acid sequence determination of proteins eluted from gels, p27.5 was identified as J chain and p16 as the C terminal fragment of mu-chain. The synthesis of two other proteins, 13 kd and 18 kd in size, was elevated in NZB spleen lymphocytes. The 18 kd protein was identified as translation initiation factor eIf-4D. The increased level of this protein may be related to the upregulation of immunoglobulin synthesis.     

Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of (32)P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem.250, 4007-4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of (32)P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t((1/2))=5-12s at 25 degrees C. Between 40 and 70% of the (32)P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function.     

Purification and identification of a CD25 expression inhibitory protein from cell-free supernatants of chronically HIV-infected promonocytic cells.

The CD25 (IL-2-R alpha) cell surface glycoprotein expressed transiently during T-cell activation is implicated in the high affinity IL-2 receptor. This paper shows that cell-free supernatants from chronically HIV-infected promonocytic cells spontaneously produce a soluble factor which inhibits CD25 expression on PHA-activated human PBMC. We purified the CD25 expression inhibitory activity by a factor 12,350, using XM50 ultrafiltration, Superose 12 molecular sieving chromatography and MonoQ anion-exchange chromatography. Then we associated this activity to one single spot (M(r) 29,000, pI 6.8) on an O'Farrell two-dimensional gel. Our data demonstrate that this protein (M(r) 29,000, pI 6.8) is released from HIV-infected promonocytic cells and suggest that this factor is a new monokine regulating the T-cell activation process.     

Mineralocorticoid-specificity of aldosterone-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders.

We have identified a group of proteins (Mr approximately 70000-80000; pI approximately 5.8-6.4) in giant-toad (Bufo marinus) urinary-bladder epithelial cells whose synthesis appears to be related to aldosterone-stimulated Na+ transport. To define this relationship further, we examined whether submaximal natriferic concentrations of aldosterone induced these proteins and whether spironolactone (a specific mineralocorticoid antagonist in renal epithelia) inhibited their synthesis. Short-circuit current was used to measure Na+ transport and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. Submaximal natriferic concentrations of aldosterone (1.4 X 10(-8) M) induced the same proteins as maximal concentrations of the hormone (1.4 X 10(-7) M). In contrast, in previous experiments, similar proteins were not induced by subnatriferic concentrations (5.0 X 10(-8) M) of cortisol, a glucocorticoid. A spironolactone/aldosterone molar ratio of 2000:1 was required to inhibit aldosterone-stimulated Na+ transport completely; ratios of 200:1 and 500:1 produced partial inhibition. Concentrations of spironolactone that abolished aldosterone-stimulated Na+ transport also inhibited aldosterone-induced protein synthesis. We conclude that the synthesis of the proteins we have identified is specifically related to activation of the mineralocorticoid pathway.     

Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells. Cells were stimulated with different steroidogenic compounds in the presence of [32P]orthophosphoric acid for 2 h and phosphoproteins analyzed by two-dimensional polyacrylamide gel-electrophoresis (PAGE). Results demonstrated an increase in the phosphorylation of four proteins (22 kDa, pI 5.9; 24 kDa, pI 6.7 and 30 kDa, pI 6.3 and 6.5) in response to 34 ng/ml hCG, 1 mM dbcAMP and 100 microM forskolin. Conversely, treatment of cells with PMA increased the phosphorylation of only one of these proteins (30 kDa, pI 6.3). At least two of these proteins (30 kDa, pI 6.5 and 6.3) appear to be identical to proteins which we and others have shown to be synthesized in response to trophic hormone stimulation in adrenal, luteal and Leydig cells. In addition, they also appear to be identical to adrenal cell mitochondrial proteins demonstrated to be phosphorylated in response to ACTH. These data indicate that proteins similar to those phosphorylated in adrenal cells in response to ACTH are phosphorylated in hormone stimulated testicular Leydig cells and that these proteins may be involved in steroidogenic regulation.     

Spermine induced phosphorylation of a 42 kD/pI 5.9 nuclear protein in different human tumor cell lines

In vitro phosphorylation of 5 M urea extracts from nuclei obtained from different human tumor cell lines leads to incorporation of phosphate from 32P-gamma-ATP in more than 20 polypeptides with an acidic pI. Whereas heparin at a concentration of 1 microgram had no effect on the phosphorylation pattern, spermine stimulated the total phosphorylation up to twofold. Furthermore, in the presence of this polyamine, the two-dimensional polyacrylamide gel revealed an additional phosphoprotein with an apparent pI of 5.9 and a relative molecular mass of 42 000. Phosphoamino acid analysis of the most prominent phosphoproteins showed serine and threonine as phosphoacceptors.     

Effects of retinoids on human bronchial epithelial cells: differential regulation of hyaluronate synthesis and keratin protein synthesis

Respiratory tract epithelia are one type of tissue targeted by vitamin A. In this study the effects of vitamin A and its analogs (retinoids) on human bronchial epithelial (HBE) cells have been investigated in a serum-free hormone-supplemented medium. This serum-free medium, which was developed for the long-term cultivation of protease-dissociated HBE cells, consists of Ham's F12 nutrient medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, and bovine hypothalamus extract. Under these in vitro conditions, retinoids specifically stimulate the synthesis and secretion of hyaluronate (HA) and alter the pattern of synthesis of keratin proteins. In regard to HA, the degree of stimulation ranges from two-fold to ten-fold and is concentration dependent. In regard to keratin proteins, the most prominent effects of retinoids are inhibition of synthesis of the 48 kd and 50 kd keratin proteins (corresponding to cytokeratins 16 and 14, respectively, in the catalog of human cytokeratins; Moll et al., 1982) and stimulation of synthesis of the 40 kd and 52-54 kd proteins. The data indicate that retinoid effects on HA and keratin protein synthesis occur at different levels. The stimulation of HA synthesis occurs immediately after the addition of retinoid and cannot be prevented by pretreatment with actinomycin D, whereas the alterations in the pattern of keratin protein synthesis appear later and are inhibited by treatment with actinomycin D at or before the administration of retinoid. This study demonstrates that HBE cultures maintained in the serum-free condition can serve as an in vitro model to elucidate the mechanisms of retinoid actions.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

A basic cytoplasmic protein (p27) induced by serum in growth-arrested 3T3 cells but constitutively expressed in primary fibroblasts.

Serum stimulation of quiescent 3T3 cells immediately induces the synthesis of a set of basic proteins that are absent in growing cells. The induction of some of these polypeptides p27 (27 kd), p35 (35 kd), p38 (38 kd) and p69 (69 kd) can be 'superinduced' in the presence of cycloheximide and completely blocked by actinomycin D. In vitro translation experiments show that the levels of mRNA coding for these proteins in serum-stimulated cells are several fold higher than in non-stimulated cells. Induction of p35 and p38 is transient (4 h); in contrast, p27 and p69 are induced for a longer period (8 h). Platelet-derived growth factor and fibroblast growth factor strongly induce p35 and p69 but weakly induce p27 and p38. Cultures of primary mouse fibroblasts express p27 but not the other polypeptides at levels similar to those found in serum-stimulated quiescent 3T3 cells. Enucleation and Triton extraction of cells show that p27 is a soluble cytoplasmic protein. The synthesis of this protein in density-arrested or serum-deprived primary cultures is only 20% reduced showing that the expression of p27 in these cells is independent of cell proliferation.     

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.     

Two-dimensional analysis of proteins phosphorylated in E. coli cells

Proteins phosphorylated in Escherichia coli cells were analyzed by the O'Farrell two-dimensional gel technique. Cytoplasmic and ribosomal fractions were studied separately. Double labeling with [32P]orthophosphate and [35S]sulfate followed by selective autoradiographic detection of each radioisotope allowed precise location of 12 major phosphoproteins on the total protein pattern of bacteria. Both the molecular mass and isoelectric point of these phosphoproteins were determined.