Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.     

Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells

In an attempt to elucidate the intracellular events regulating the proliferation of endothelial cells (EC), we have compared the phosphorylation events in membranes prepared from proliferating (sparse) and quiescent (confluent) EC. Triton-solubilized membranes from sparse and confluent EC were incubated at pH 6.5 in the presence of divalent cations and [32P]ATP. Membrane proteins were then separated by SDS-PAGE and the radiolabeled phosphoproteins visualized by autoradiography. The overall kinase activity per milligram protein was 1.7 +/- 0.2-fold greater in membranes prepared from proliferating than from quiescent cells. The extent of phosphorylation was dramatically elevated in sparse over confluent samples for four phosphoproteins having the following approximate molecular masses: 180, 100, 97, and 55 kDa. The 180 and 100 kDa phosphoproteins exhibited 3.6- and 7.4-fold higher labeling, respectively, in sparse than in confluent membranes and both were phosphorylated on serine residues exclusively. The 97 kDa phosphoprotein was 11.6-fold higher in sparse membranes and contained both phosphoserine (p-ser) and phosphotheronine (p-thr), the latter comprising 61% of the radioactivity. The 55 kDA phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr) and was 2.3-fold higher in sparse membranes. Of these four phosphoproteins, only the 55 kDa protein was phosphorylated in confluent samples to an appreciable degree. Whereas the p-ser and p-thr content of the 55 kDa band increased moderately in sparse vs. confluent sample (1.8-fold increase), the tyrosine residues of this protein in sparse membranes were radiolabeled to a much greater extent relative to confluent membranes (5.4-fold increase). Analysis of the cofactor requirements of the FC membrane kinase(s) revealed that Mn2+ is the optimum cofactor and that Mg2+ can replace Mn2+ only for the kinase acting on the 100 kDa band. This suggests the presence of multiple EC membrane kinases. In the presence of both cofactors, the phosphorylation pattern is similar to the pattern obtained with Mn2+ alone. The kinase activity acting on all four phosphoproteins was independent of Ca2+, cAMP, cGMP, and phorbol 12-myristate 13-acetate. The mechanism responsible for the difference in kinase activity of proliferating vs. quiescent cells was not due to an inhibitor or enhanced phosphatase activity in confluent cells; the phosphorylation patterns obtained with sparse solubilized membranes and a mixture of sparse and confluent solubilized membranes were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Defective sarcolemmal phosphorylation associated with noninsulin-dependent diabetes

Noninsulin-dependent diabetes is associated with a decrease in the activity of sarcolemmal phosphatase 1, but no change in the activities of phosphatase 2A, 2B, or 2C. Also unaffected by diabetes were the activities of protein kinase C, cAMP-dependent protein kinase and calcium-calmodulin protein kinase. Because of the decrease in phosphatase 1 activity, 32P incorporation into sarcolemmal phosphoproteins catalyzed by either intrinsic protein kinases or extrinsic cAMP-dependent protein kinase was elevated in the diabetic. Among the proteins whose phosphorylation was elevated in diabetes was the phospholamban-like protein, which has been implicated in the regulation of ATP-dependent calcium transport. The phosphate-linked increase could be prevented by exposing the membranes to a phosphatase inhibitor and either extrinsic cAMP-dependent protein kinase or alamethicin. In addition to the phosphatase-linked effects, analysis of individual sarcolemmal phosphoproteins by SDS-polyacrylamide gel electrophoresis indicated that diabetes caused a specific elevation in membrane phosphorylation of some proteins (43 kDa and 78 kDa), but a decrease in the phosphorylation state of other phosphoproteins (31 kDa and 49 kDa). The data indicate that membrane phosphorylation is dramatically altered by diabetes. The possibility that this contributes to altered myocardial function is discussed.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Phosphorylation of isolated plasma membranes of AH-66 hepatoma ascites cells by casein kinase 1

The isolated plasma membranes of AH-66 hepatoma cells were phosphorylated by casein kinase 1 purified from the cytosol fraction of AH-66 cells. Casein kinase 2 purified from the same source had little effect on the phosphorylation of the plasma membranes. Two-dimensional gel electrophoresis and autoradiography showed that casein kinase 1 enhanced the phosphorylation of approx. 10 plasma membrane proteins that are phosphorylated only faintly in the isolated plasma membranes by endogenous protein kinase. Among these phosphoproteins, tubulin was identified as judged from their molecular weights and isoelectric points. These results suggest that one of the physiological functions of casein kinase 1 is phosphorylation of plasma membrane and plasma membrane-associated proteins.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Interleukin 2 stimulates tyrosine phosphorylation in T cell membrane fractions

Interleukin 2 is a growth factor secreted by T lymphocytes upon antigenic stimulation and inducing the proliferation of T cells bearing at their surface the heterodimeric high-affinity form of its receptor. No enzymatic function has so far been demonstrated in the receptor subunits. In an attempt to elucidate the biochemical pathway of signal transduction, we investigated the capacity of interleukin 2 to modulate tyrosine phosphorylation in T cell membranes. Membrane-rich fractions from T cells were tested for their ability to phosphorylate tyrosine in the presence or absence of added recombinant interleukin 2. Using as substrate a synthetic polymer of glutamic acid and tyrosine, we demonstrated a 3-4-fold stimulation of tyrosine phosphorylation in the presence of interleukin 2; this stimulating effect appeared to be well correlated with interleukin 2 function since (a) it was not observed in insensitive cells, (b) it required the presence of the high-affinity form of the receptor and (c) it was dose-dependent. Confirmatory results were obtained by phosphorylating membrane-rich fractions with [gamma-32P]ATP and by analysing the resulting phosphoproteins: only in fractions from cells with the high-affinity form of the receptor were several membrane proteins specifically phosphorylated on tyrosine residues in response to interleukin 2. At least two proteins of 115 and 58 kDa were consistently hyperphosphorylated on tyrosine in an interleukin-2-dependent manner. This stimulation was strongly dependent on the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate. Thus, we propose that interleukin 2 enhances tyrosine phosphorylation by stimulating a tyrosine kinase activity. The nature of the enzyme involved remains to be determined.     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

Activation of a membrane-associated phosphatidylinositol kinase through tyrosine-protein phosphorylation by naphthoquinones and orthovanadate.

We have previously reported that several naphthoquinones stimulated tyrosine-specific protein phosphorylation in isolated rat liver membranes. Our more recent study demonstrated a similar effect by orthovanadate, which concomitantly stimulated phosphorylation of protein-tyrosine and phosphatidylinositol (Ptd-Ins). Results presented here show a simultaneous increase in PtdIns phosphorylation along with stimulation of tyrosine-protein phosphorylation by naphthoquinones. This PtdIns kinase resembles the type I PtdIns kinase in that it was insensitive to adenosine inhibition. The product, nevertheless, comigrated with a PtdIns-4-phosphate standard in TLC using three different solvent systems. Stimulation of PtdIns phosphorylation by vanadate or naphthoquinones could be achieved in the following preparations: intact rat liver membranes, Triton X-100-solubilized membranes, solubilized membranes partially purified by Sephacryl chromatography, solubilized membranes purified by wheat germ agglutinin chromatography. The naphthoquinone or vanadate-activated PtdIns kinase activity could be isolated by antiphosphotyrosine antibody-agarose affinity chromatography. The relative potencies of a series of ring-substituted naphthoquinones in the stimulation of tyrosine-protein phosphorylation, PtdIns kinase activity, dithiothreitol-dependent oxygen consumption, and cytochrome c reduction were highly correlated. We conclude that oxidant(s) produced by redox cycling of naphthoquinones stimulated an adenosine-insensitive PtdIns kinase through tyrosine phosphorylation of the enzyme.     

Two alternative mechanisms control the interconversion of functional states of the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha bind to a common receptor at the cell surface. Both the affinity and the tyrosine protein kinase activity of the receptor are regulated by exogenous factors, such as platelet-derived growth factor. A protein kinase C-dependent (Ca2+/phospholipid-dependent enzyme) and independent regulatory mechanism have been described. The protein kinase C-dependent mechanism results in the inhibition of the affinity and tyrosine kinase activity of the EGF receptor. We describe in this report an alternative mechanism of regulation of the receptor that is mediated by sphingosine. Treatment of WI-38 human fetal lung fibroblasts with 5 microM sphingosine for 2 min at 37 degrees C caused a marked increase in the affinity of the EGF receptor. Similar results were obtained when isolated plasma membranes prepared from these cells were incubated with sphingosine. A stimulation of the EGF receptor tyrosine protein kinase activity was also observed after sphingosine-treatment of plasma membranes. Sphingosine caused a decrease in the Km for ATP and an increase in the Vmax for the tyrosine phosphorylation of a synthetic peptide substrate. Control experiments demonstrated that these actions of sphingosine were not secondary to the inhibition of protein kinase C. These data indicate that sphingosine causes the functional conversion of the EGF receptor into an activated state that expresses both a high affinity for EGF and an increased tyrosine kinase activity. We conclude that sphingosine is a bioactive molecule in human fibroblasts.     

Association of the Transformation-Specific Protein pp60src with the Membrane of an Avian Sarcoma Virus

The transformation-specific protein pp60(src) coded for by avian sarcoma viruses and its associated protein kinase activity is present in virus particles of Rous sarcoma virus, Schmidt-Ruppin strain, subgroup D. Quantitative comparison of the immunoglobulin G-phosphorylating activity in Schmidt-Ruppin D virus and Schmidt-Ruppin D virus-transformed fibroblasts indicated that there was two- to fourfold less activity in the virus particles. Disruption of virus particles with nonionic detergent demonstrated that the protein kinase activity fractionated together with the viral membrane protein gp85. Therefore, viral membranes were isolated by floating detergent-disrupted virus through a discontinuous sucrose density gradient. At a characteristic density corresponding to 26% sucrose, viral membranes were identified by the radioactively labeled viral glycoprotein and furthermore by the membrane marker enzyme Na(+)-K(+)-stimulated, Mg(2+)-activated ATPase and were visualized by electron microscopy. Contamination by cell membranes could be ruled out, since (i) the virus preparation was free of cell membrane contaminants as judged from electron microscopy, (ii) floating of intact virus did not release membraneous material, and (iii) virus-free tissue culture fluid from Schmidt-Ruppin D virus-transformed nonproducer cells (which potentially contain cell membranes) did not contribute any immunoglobulin G-phosphorylating activity after mixing with nontransforming virus and pelleting it. Both pp60(src) and the protein kinase activity were found to be associated with the viral membrane. Solubilization of virus by detergent released two phosphoproteins, with molecular weights of 42,000 and 45,000 which reacted with sera specific for pp60(src) and revealed protein kinase activity but which were not membrane bound and may have represented degradation products of pp60(src). Surface iodination of intact virus particles (harvested at 3-h intervals) did not result in radioactive labeling of pp60(src), whereas collection at 24-h intervals allowed iodination of pp60(src). In contrast to the viral glycoprotein gp85, the iodinated virion-associated pp60(src) was insensitive to mild proteolytic treatment. Binding to tumorbearing-rabbit serum, immunoglobulin G phosphorylation, and endogenous phosphorylation of 60,000-, 45,000-and 42,000-dalton proteins required lysed virus and were not possible with intact virus. These results indicated that pp60(src) was embedded within the viral membrane. Membrane proteins phosphorylated in vitro were analyzed for their phosphoamino acid composition. Eight polypeptides exhibited phosphorylation in tyrosine and were absent in nontransforming viral controls.     

Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages.

Bacterial lipopolysaccharide (LPS) is a potent activator of antibacterial responses by macrophages. Following LPS stimulation, the tyrosine phosphorylation of several proteins is rapidly increased in macrophages, and this event appears to mediate some responses to LPS. We now report that two of these tyrosine phosphoproteins of 41 and 44 kDa are isoforms of mitogen-activated protein (MAP) kinase. Each of these proteins was reactive with anti-MAP kinase antibodies and comigrated with MAP kinase activity in fractions eluted from a MonoQ anion-exchange column. Following LPS stimulation, column fractions containing the tyrosine phosphorylated forms of p41 and p44 exhibited increased MAP kinase activity. Inhibition of LPS-induced tyrosine phosphorylation of these proteins was accompanied by inhibition of MAP kinase activity. Additionally, induction of p41/p44 tyrosine phosphorylation and MAP kinase activity by LPS appeared to be independent of activation of protein kinase C, even though phorbol esters also induced these responses. These results demonstrate that LPS induces the tyrosine phosphorylation and activation of at least two MAP kinase isozymes. Since MAP kinases appear to modulate cellular processes in response to extracellular signals, these kinases may be important targets for LPS action in macrophages.     

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.     

Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases.

Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins. The 44 kd protein was immunoprecipitated with an antibody directed against extracellular signal-regulated kinase 1/mitogen-activated protein kinase (MAPK) and the 42 kd protein comigrated with a 42 kd MAPK, indicating that at least one and probably both Ras-regulated phosphoproteins are MAPKs. In addition, MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, was also regulated by Ras. Ras was not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs.     

Hyaluronic acid stimulates protein kinase activity in intact cells and in an isolated protein complex

The addition of hyaluronate to intact chick embryonic heart fibroblasts enriched with a hyaluronate-binding protein (HABP) stimulated phosphorylation of tyrosine and serine/threonine residues in cellular proteins. A protein complex containing a hyaluronate-binding protein (cell-HABP) was isolated from the cultured heart fibroblasts. The isolated complex (Mr approximately 1 x 10(6] contained phosphoproteins that exhibited protein kinase activity specifically stimulated by hyaluronate. Both tyrosine and serine residues in the protein complex were phosphorylated in response to this glycosaminoglycan. The hyaluronate-stimulated protein kinase activity was tightly associated with cell-HABP in vitro; enzyme activity co-immunoprecipitated with cell-HABP using a monospecific anti-HABP antibody and co-eluted with cell-HABP when chromatographed on a column of Sephacryl S-1000 in 2.0 M guanidine hydrochloride. The uniqueness of the cell-HABP-associated protein kinase activity was suggested by both its specific response to hyaluronate, relative to related glycosaminoglycans such as heparin and chondroitin sulfate or to growth factors such as epidermal growth factor or insulin, and its antigenic distinction from other protein kinases such as growth factor receptors. These results point to a new mechanism by which glycosaminoglycans, such as hyaluronate, may modify cell behavior.     

Phosphorylation of thylakoid proteins by a purified kinase

A simplified method is given for the purification of a 64-kilodalton protein kinase from spinach or pea thylakoid membranes (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385). In a heterogeneous reconstitution system comprised of purified kinase and washed thylakoids (having their intrinsic kinase inactivated or removed), endogenous light-harvesting pigment protein of photosystem II could serve as a substrate. Its phosphorylation did not require rebinding of kinase to the thylakoid membrane and, like the phosphorylation of solubilized pigment protein, was not under redox control. No reconstitution was observed upon replacing 64-kilodalton protein kinase with 25-kilodalton protein kinase (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 14062-14068). Tryptic digestion of phosphorylated membranes removed the site of phosphorylation; the phosphorylated amino acid present in light-harvesting pigment protein and its tryptic peptide was threonine. Immunoglobulin from a polyclonal antiserum, raised against the purified enzyme, fully inhibited kinase activity toward solubilized and endogenous pigment protein. At higher titers, the antibody was effective in totally inhibiting the redox-sensitive phosphorylation of thylakoid proteins by endogenous kinase; inhibition profiles for phosphorylation of pigment protein and thylakoid proteins of 32, 16, and 9 kilodaltons were essentially identical. The 64-kilodalton protein kinase would thus appear to be responsible for all of the observed phosphorylation of thylakoid phosphoproteins.     

Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.     

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.     

Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.     

Granulocyte-macrophage colony-stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor-dependent hematopoietic cell line.

Steel factor (SF), the ligand for the proto-oncogene c-kit, acts synergistically with GM-CSF or IL-3 to support the growth of normal human hematopoietic progenitor cells. We examined the effects of SF on GM-CSF or IL-3 induced proliferation of a human factor-dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL-3 or GM-CSF alone, and its addition dramatically enhanced (three- to sixfold) maximal GM-CSF or IL-3 stimulated proliferation. SF did not increase the number or affinity of cell surface GM-CSF receptors. We examined several early events of signal transduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinase, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM-CSF and IL-3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140-150, 116, 106, 94, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal transduction intermediates known to be phosphorylated and activated by GM-CSF and IL-3, the 70-75 kDa Raf-1 kinase, and p42 mitogen-activated protein kinase-2 (MAPK), were also phosphorylated by SF. Combinations of GM-CSF or IL-3 with SF did not further increase the phosphorylation of Raf-1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM-CSF or IL-3, induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). These results indicate that SF and GM-CSF/IL-3 have partially overlapping effects on early signal transducing events, as well as striking differences, such as tyrosine phosphorylation of PLC-gamma. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.