Abstract: Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating specific endogenous membrane proteins, as well as exogenously added histone. The binding of EGF to membranes in vitro resulted in a several-fold stimulation of the phosphorylation reaction. The phosphorylation reaction was not dependent on cyclic AMP or cyclic GMP. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system. The activation of the membrane-associated protein kinase by EGF appears to be a reversible phenomenon. The membrane preparation could be solubilized by a number of non-ionic detergents with the retention of both 125I-labeled EGF binding activity and EGF-enhanced phosphorylation of specific membrane proteins. The solubilized membrane preparation was purified by affinity chromatography. The purified preparation retained both EGF-binding activity and EGF-enhanced phosphorylation activity. Analysis of the affinity-purified preparation by SDS gel electrophoresis indicated the presence of one major protein band of molecular weight 150,000 and several trace bands. The evidence suggests that the major 150,000 protein band is the receptor for EGF and is a substrate of the phosphorylation reaction. The co-purification of EGF-binding activity and EGF-stimulated phosphorylation activity suggests an inherent close relationship. The EGF-stimulated phosphorylation reaction appears to be specific for tyrosine residues and in this regard resembles the src protein kinase.

Abstract: Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.

Abstract: Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.

Abstract: Potential antiviral nucleoside analogs 1-beta-D-arabinofuranosylthymine, the 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-nucleosides of -5-methyluracil, -5-iodouracil, -5-methylcytosine, -5-iodocytosine, and -E-5-(2-bromovinyl)uracil, E-5-(2-bromovinyl)-2'-deoxyuridine, E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil, and 9-(2-hydroxyethyoxymethyl)guanine were studied to compare their phosphorylation rates relative to thymidine by purified thymidine kinases from human and herpes simplex virus sources. Most of these analogs are capable of being phosphorylated by both human and viral enzymes. On the assumption that inhibition constants (Ki) reflect binding affinity, Ki values were determined for these analogs with the same thymidine kinases. In general, these analogs have a greater affinity for the viral enzymes. The amount of the analogs phosphorylated to the monophosphate form, which is presumably necessary to produce cytotoxic effects, was determined by the combined effects of phosphorylation rates and binding affinities. All of these analogs act as preferential substrates for the viral thymidine kinases at low concentrations, which may be one of the main reasons for their selective antiviral action.

Abstract: Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.

Abstract: Dichloroacetate activates the pyruvate dehydrogenase complex of many tissues by inhibiting the kinase responsible for phosphorylation and inactivation of the complex. Dichloroacetate also activates the myocardial branched-chain α-keto acid dehydrogenase complex but apparently not by direct inhibition of the analogous kinase. Oxalate and glyoxylate, metabolites of dichloroacetate, are responsible for some in vitro effects of dichloroacetate. Dichloroacetate stimulates leucine oxidation by isolated hepatocytes because glyoxylate transaminates with leucine. Dichloroacetate inhibits lactate gluconeogenesis by hepatocytes incubated in low bicarbonate buffer because oxalate inhibits pyruvate carboxylase under such conditions. In vivo, dichloroacetate decreases blood glucose by limiting the supply of gluconeogenic precursors to the liver. This effect is a consequence of pyruvate dehydrogenase activation in peripheral tissues. Dichloroacetate lowers blood cholesterol in hyperlipidemic patients by uncertain means. Dichloroacetate has been tried experimentally in treatment of diabetes, hypercholesterolemia, and hyperlactatemia, but it has neurotoxicity, can cause cataracts, and may be mutagenic.

Abstract: Ca2+ plays a major role in the functional use of tubulin in brain and other tissues. It activates an endogenous tubulin kinase system in brain cytosol, tubulin, and presynaptic nerve terminal fractions prepared from rat brain. Activation of the Ca2+ tubulin kinase system was modulated by the Ca2+ receptor protein calmodulin. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.4 micrograms, respectively. Ca2+ -calmodulin tubulin kinase activity was very unstable after death, and procedures were developed to stabilize the activity of this enzyme system. Evidence is presented demonstrating that the Ca2+ -calmodulin tubulin kinase system is distinct from the previously described cyclic AMP-Mg2+ tubulin kinase. The results suggest that Ca2+- and calmodulin-stimulated phosphorylation of tubulin may be a major biochemical mechanism modulating some of calcium's effects on tubulin and may play a significant role in mediating some of calcium's actions on cell functions.

Abstract: The product of the Rous sarcoma virus (RSV) transforming gene, src, is a phosphoprotein of molecular weight (Mr) 60,000 (pp60src)1–3 that is responsible for cell transformation as well as fibrosarcoma formation in a variety of animals4,5. Several experiments suggest that pp60src is a protein kinase6–10 with the unusual capacity of phosphorylating tyrosine residues11,12, but additional evidence would be of value. Earlier studies had suggested that bacteria were unable to carry out protein phosphorylation13, although more recent findings indicate that distinct protein kinases and phosphatases are present in Salmonella typhimurium14. Despite this observation, because of the evolutionary distance, the enzymatic activities observed in bacteria are likely to be carried out by proteins unrelated to those expressed in eukaryotic cells. Thus, to study the RSV transforming protein synthesized in the absence of the variety of protein kinases expressed in normal host cells (for reviews see refs 15, 16), we have now constructed plasmids that express p60src in Escherichia coli. Analysis of p60src produced in E. coli and selected by immunoaffinity chromatography, shows that it has the capacity to phosphorylate proteins at tyrosine residues; this activity is specifically inhibited by anti-p60src IgG. Extracts from E. coli carrying identical plasmids but lacking the src gene yield no detectable enzyme activity. These data, taken with those previously published9,10,17, lead to the conclusion that the RSV src gene encodes a protein kinase.

Abstract: The histone 1 (H1) fraction of HeLa S-3 cells contains two principal subtypes, H1A (Mr approximately 21 000) and H1B (Mr approximately 22 000). In G1 cells, the H1 molecules are distributed among several phosphorylation states, most H1A molecules containing 0 or 1 phosphate groups and most H1B molecules containing 0, 1, 2, or 3 phosphate groups. Both subtypes undergo a general increase in phosphorylation levels of approximately 1 P/mol during the S phase and a further increase or 3--4 P/mol during mitosis. These two increases affect most of the H1 molecules and thus reflect phosphorylations occurring widely throughout the chromatin, presumably in association with replication and mitotic chromosome condensation. During all these periods, multiple phosphorylation levels of H1 molecules persist, as does the phosphorylation differential between H1A and H1B. Thus, there appear to be phosphorylation states that only some of the H1 molecules occupy, a fact that may be related to the conformational diversity in interphase and mitotic chromatin. The existence of differences between H1A and H1B phosphorylation states throughout the cell cycle, and within a single cell type, is in accord with the hypothesis that the H1 subtypes are functionally distinct, such that subtype-specific phosphorylations contribute to the control of chromatin organization.

Abstract: Both lymphocytes and fibroblasts that have been transformed by ABelson murine leukemia virus contain 6- to 12-fold increased levels of the rare modified amino acid phosphotyrosine in their proteins. This observation, coupled with the fact that the p120 protein encoded by this virus has been shown to undergo an apparent autophosphorylation to yield phosphotyrosine in vitro, suggests that Abelson virus encodes a protein kinase that phosphorylates tyrosine in transformed cells. These results are similar to those obtained previously with Rous sarcoma virus and suggest, by analogy, that the modification of cellular polypeptides through the phosphorylation of tyrosine may be involved in cellular transformation by Abelson virus. p120 isolated from transformed cells contains phosphoserine, phosphothreonine, and phosphotyrosine. The phosphotyrosine is found at two sites in the protein. p120 therefore may be a protein kinase that undergoes autophosphorylation in vivo.

Abstract: We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000-dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.

Abstract: In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains. In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared. According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis. (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II. This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII). (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correspondence. (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement, (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II. It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical. It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.

Abstract: Enhanced phosphorylation of two specific protein bands accompanied catecholamine secretion from cultured bovine adrenal medulla cells stimulated by different secretagogues. Cells preincubated with 32Pi were treated with nicotine, veratridine, Ionomycin, or barium. Each of these secretagogues stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 95,000. Phosphorylation of the 60,000 M.W. protein band was two- to threefold higher than that of the 95,000 M.W. band on stimulation with nicotine, veratridine, or barium, but Ionomycin stimulated phosphorylation of each protein band to the same extent. In general, the increase in phosphorylation was most rapid during the first minute of stimulation and occurred prior to detectable secretion. Phosphorylation reached a relatively constant level within 5 min after onset of stimulation at a time when catecholamine release was still proceeding at a rapid rate. Nicotine-stimulated phosphorylation and catecholamine secretion were calcium-dependent and blocked by d-tubocurarine, whereas tetrodotoxin inhibited veratridine-stimulated secretion and phosphorylation. We conclude that catecholamine secretion and protein phosphorylation occur under similar conditions and that Ca2+-dependent incorporation of phosphate into specific proteins may be a link in stimulus-secretion coupling.

Abstract: Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.

Abstract: Two key steps in meiotic maturation of the Xenopus oocyte involve protein phosphorylation–dephosphorylation1. A burst of cyclic AMP-independent phosphorylation occurs at the time of the breakdown of the nuclear envelope, whether maturation is triggered by progesterone, by the inhibitor of cyclic AMP-dependent protein kinase (PKI)2 or by the maturation promoting factor (MPF)1,3. Also, an in ovo decrease in the level of the free catalytic (C) subunit of cyclic AMP-dependent protein kinase, induced by microinjection of pure PKI4, initiates meiotic maturation1,2,4. This suggests that MPF appears only after dephosphorylation of a phosphorylated maturation protein Mp-P4. We now show, by microinjection of pure inhibitor-1 of protein phosphatase-15, which blocks progesterone- and PKI-induced maturation but not the MPF-induced one, that protein phosphatase-1 catalyses the dephosphorylation of Mp-P.

Abstract: We have previously shown that fluorescein 5′ isothiocyanate is a potent inhibitor of the Ca-ATPase from sarcoplasmic reticulum [Pick, U. and Karlish, S. J. D. (1980) Biochim. Biophys. Acta, 626, 255–261] and completely blocks Ca uptake with ATP but not with acetyl phosphate [Pick, U. and Bassilian, S. (1981) FEBS Lett. 123, 127–130]. Here we show that fluorescein isothiocyanate inhibits in parallel all the ATP-dependent partial reactions: the Ca-ATPase, ATP-dependent Ca uptake and phosphorylation by ATP whereas the phosphorylation reaction by inorganic phosphate is resistant to fluorescein isothiocyanate and Ca uptake with acetyl phosphate is only partly inhibited by the modification. The time course of inactivation by fluorescein isothiocyanate is biphasic and the rate of inactivation is markedly increased at alkaline pH. The observations that a lysine-specific reagent (2,4,6-trinitrobenzene sulfonic acid) com- petes with binding of fluorescein isothiocyanate and the pH dependence of inactivation strongly suggests binding to a lysine E-amino group. Mg ions accelerate the rate of inactivation at pH 7.5–8.0 and Ca ions slightly inhibit the inactivation and the binding of fluorescein isothiocyanate. The adenine nucleotides ATP and AdoPP [NH]P protected against inactivation by fluorescein isothiocyanate. A kinetic analysis of the initial rates of inactivation indicates competition between ATP and fluorescein isothiocyanate (the apparent dissociation constant KATP= 38 μM) and suggests a common binding site. Ca ions increase the apparent affinity of the non-hydrolyzable analog AdoPP [NH]P by an order of magnitude but have no appreciable effect on the apparent affinity of ATP. Mg ions decrease the apparent affinity for adenine nucleotides both in the presence and absence of Ca. It is suggested that (a) fluorescein isothiocyanate binds to and modifies the ATP-binding site of the enzyme. (b) The enzyme has only one ATP-binding site but the site exists in two different configurations which sequentially appear at the E2 and the El conformations of the enzymes; and correspond to the ‘regulatory’ ATP-binding site and to the phosphorylation site and (c) the site is blocked by fluorescein isothiocyanate in both configurations.

Abstract: At least two substrate proteins for phospholipid-sensitive Ca2+-dependent protein kinase and at least six substrates for calmodulin-sensitive Ca2+-dependent protein kinase were identified in the cytosol of the guinea pig heart. In the particulate subfractions enriched in nuclei, mitochondria, microsome, or plasma membrane, no substrates for the phospholipid-sensitive enzyme were demonstrated but at least four substrates for the calmodulin-sensitive enzyme were identified. The present studies suggest that phospholipid, acting independently of calmodulin, is likely to be involved in the regulation of Ca2+-dependent protein phosphorylation in the heart. Phosphorylation of endogenous substrates for the two enzyme systems was effectively inhibited by palmitoylcarnitine. When histone was used as exogenous substrate, the carnitine ester inhibited the cardiac phospholipid-sensitive Ca2+-dependent protein kinase but not the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It is suggested that inhibition of the Ca2+-dependent phosphorylation of cardiac proteins, regulated by either phospholipid or calmodulin, is probably related in part to the great increase in this fatty acid metabolic intermediate in the ischemic heart.

Abstract: Protein phosphorylation was examined in whole cell extracts from normal and avian sarcoma virus-transformed chicken embryo fibroblasts. The addition of serum or epidermal growth factor to serum-starved normal cells resulted in increased 32P labeling of a Mr 30,000 protein. In extracts from cells transformed by a temperature-sensitive mutant of Schmidt-Ruppin virus, subgroup A, and grown at the permissive temperature, the protein was phosphorylated regardless of serum starvation. This Mr 30,000 protein was shown to be ribosomal protein S6, and the effects of avian sarcoma virus transformation on S6 phosphorylation were further investigated. The ability to phosphorylate S6 in the absence of serum was found to be temperature sensitive when S6 preparations from the temperature-sensitive mutant-infected cells incubated at permissive and nonpermissive temperatures were compared. Cells transformed by the parent virus (Schmidt-Ruppin, subgroup A) maintained the ability to phosphorylate S6 in the absence of serum when incubated at either temperature. Phosphoserine was the only phospho-amino acid detected in acid hydrolysates from phosphorylated S6 preparations.