Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.     

Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators

Recently, we reported the purification of a 46-kDa membrane-associated platelet protein which is phosphorylated in intact platelets and platelet membranes by cGMP- and cAMP-dependent protein kinases (Halbrügge, M., and Walter, U. (1989) Eur. J. Biochem. 185, 41-50). Here we demonstrate that both cGMP- and cAMP-dependent protein kinases catalyze the rapid incorporation of up to 1.4 mol of phosphate/mol of this purified vasodilator-stimulated phosphoprotein (VASP). A specific rabbit antiserum was prepared which recognized both the 46-kDa dephospho form and the 50-kDa phospho form of VASP in Western blots. In untreated washed platelets, VASP was found to be present primarily as a 46-kDa dephosphoprotein. Sodium nitroprusside (100 microM) raised the intracellular platelet cGMP concentration from approximately 0.44 to 4.1 microM, without a significant effect on the cAMP level, and converted up to 50% of VASP to the 50-kDa phospho form. Prostaglandin E1 (10 microM) raised the platelet cAMP concentration from approximately 4.4 to 28.4 microM, without a significant effect on the cGMP level, and shifted up to 67% of VASP to the 50-kDa phospho form. Removal of the vasodilators sodium nitroprusside and prostaglandin E1 from the platelet suspension was followed by a return of the cyclic nucleotide concentration to basal levels and subsequent conversion of the 50-kDa phospho form of VASP to the 46-kDa dephospho form. The results support the hypothesis that VASP phosphorylation is an important component of the intracellular mechanism of action of these vasodilators in human platelets.     

The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum

The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or 'a' form, is independent of 5'adenosine monophosphate (5'AMP) while the inactive, or 'b' form, is 5'AMP-dependent. The activity of the 'b' form predominates early in development, while the activity of the 'a' form peaks in mid-late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross-reactivity. The anti-'a' serum reacted with a 104-kDa protein that was associated with phosphorylase 'a' activity; the anti-'b' serum reacted with a 92-kDa protein that was associated with phosphorylase 'b' activity and weakly cross-reacted with the 104-kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the 'b' form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase-'a' activity and the 104-kDa protein. Immunotitration data suggested that the 'a' form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.     

Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the "linker" segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.     

Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.     

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.     

Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium

In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.     

Occurrence of two phosphorylated forms of yeast fructose-1,6-bisphosphatase with different isoelectric points

Yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) immunoprecipitated from glucose-derepressed wild-type cells and subjected to isoelectric focusing, appears as a unique peak, essentially homogeneous and devoid of incorporated phosphate. However, after cell incubation with glucose, two phosphorylated forms are detectable. The isoelectric point of one is higher and of the other is lower than that of the native form. In contrast, in the mutant ABYS1 which is deficient in several vacuolar proteinases (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343), only the more acidic phospho form appears after cell incubation with glucose. However, sequence data rule out the possibility that limited proteolysis is the event responsible for the appearance of the more basic form of the phosphoenzyme. Nevertheless, time courses of glucose-induced inactivation of fructose-1,6-bisphosphatase show that the enzyme undergoes a substantially slower inactivation in the ABYS1 mutant as compared to the wild-type. These findings point to a degradative mechanism involving, besides the well-known phosphorylation, an additional as yet unknown modification which probably sensitizes the enzyme to proteolytic attack; furthermore, the enzyme responsible for such a modification seems to require one or more of the vacuolar proteinases missing in the mutant for its maturation.     

Dipeptidyl Aminopeptidase IV from Pseudomonas sp. WO24

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS–PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus

Recent studies from this laboratory have identified novel cytoskeletal proteins that are phosphorylated on tyrosine in vivo in Rous sarcoma virus-transformed chick fibroblasts (Glenney, J. R., Jr., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408). In the present report, the phosphorylation of these proteins was examined in cells expressing the nonmyristylated mutants of src that are not transformed. A good correlation was found between transformation and the tyrosine phosphorylation of a 22-kDa protein. Tyrosine phosphorylation of the 22-kDa protein was reduced more than 95% in cells expressing the nonmyristylated mutants of src. Size fractionation revealed that the 22-kDa phosphoprotein in transformed chick fibroblasts is found in a Mr 150,000 complex. Monoclonal antibodies were used to screen various chicken tissues where the 22-kDa protein was found at high levels in muscle and lung with low levels in epithelial cells and brain. The 22-kDa protein becomes an excellent candidate for a mediator of transformation by the tyrosine kinase class of oncogenes.     

Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells

Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.     

Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells.

Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of androgen and polyamines on the phosphorylation of nucleolar proteins from rat ventral prostates with particular reference to 110-kDa phosphoprotein

The effects of testosterone (in vivo) and polyamines (in vitro) on the phosphorylation of nucleolar proteins of rat ventral prostates were studied. Phosphorylation of nucleolar proteins was accomplished by incubation of isolated nucleoli with [gamma-32P]ATP at 37 degrees C for 10 min followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Of several nucleolar phosphoproteins observed in ventral prostates of castrated rats, the incorporation of 32P into 110-kDa protein was remarkably augmented by the testosterone treatment. The stimulation became evident as early as 4 h after the injection of the hormones, reaching 3-4-fold of the control level and was efficiently prevented by cycloheximide injection 3 h before killing. 5 alpha-Dihydrotestosterone gave similar results to testosterone, but estradiol-17 beta failed to stimulate the phosphorylation of 110-kDa protein. Polyamines and cyclic nucleotides did not affect the phosphorylation, but, when phenylmethanesulfonyl fluoride was omitted from the standard medium, spermine and spermidine showed a distinct effect: 110-kDa phosphoprotein was completely abolished with a concomitant increase of 59-kDa phosphoprotein in both cases of castrated and testosterone-primed rats. The effect of polyamines seems to be due to the stimulation of degradation of the protein which is presumably catalyzed by a serine protease.     

Phosphatidate phosphatase from Saccharomyces cerevisiae. Isolation of 45- and 104-kDa forms of the enzyme that are differentially regulated by inositol

Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form of membrane-associated phosphatidate phosphatase from Saccharomyces cerevisiae (Lin, Y.-P., and Carman, G.M. (1989) J. Biol. Chem. 264, 8641-8645) revealed the existence of a 45-kDa form of the enzyme. Immunoblot analysis also showed that the 91-kDa form of the enzyme was a proteolytic product of a 104-kDa enzyme. The mitochondrial fraction contained the 45-kDa enzyme, whereas the microsomal fraction contained the 45- and 104-kDa enzymes. In vivo labeling experiments showed that the 104-kDa form of phosphatidate phosphatase was not a precursor of the 45-kDa form of the enzyme. The 45- and 104-kDa forms of phosphatidate phosphatase were purified and characterized. The enzymological properties of both enzymes were similar. However, the phosphatidate phosphatase 45- and 104-kDa proteins differed with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage. The expression of the phosphatidate phosphatase 45- and 104-kDa enzymes were regulated differentially in cells supplemented with inositol. The addition of inositol to the growth medium resulted in the induction of the phosphatidate phosphatase 45-kDa enzyme. The expression of the 104-kDa enzyme was not affected by inositol. Both forms of phosphatidate phosphatase were induced when cells entered the stationary phase of growth.     

Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.     

Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies

One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target.