Abstract: Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarization-induced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.

Abstract: Chinese hamster lung fibroblast cells (CCl39) enter the G0/G1 nonproliferative state after serum deprivation. In this report, we show that reinitiation of DNA synthesis by serum or the combination of purified human thrombin and insulin (1-10 microgram/ml) is preceded by very early stimulation of ionic fluxes (Na+/Rb+) and protein phosphorylation (27,000 daltons, 62,000 daltons, and the ribosomal S6 proteins). The potentiating action of insulin on thrombin-stimulated DNA synthesis is also observed on thrombin-stimulated Na+ influx, Rb+ influx, and protein S6 phosphorylation. Moreover, we demonstrate that CCl39 cells possess a Na+/H+ exchange system sensitive to amiloride. Half-maximal inhibition of growth factor-activated Na+ influx and Na+-dependent H+ efflux is obtained with 3-10 microM amiloride. Two lines of evidence indicate that the extrusion of H+ via the activation of the Na+/H+ exchanger is coupled to protein S6 phosphorylation: serum-stimulated phosphorylation is blocked by (i) amiloride at a concentration that abolishes serum-stimulated Na+ influx and (ii) protonophores that acidify the cell interior. The present data support the idea that the regulation of intracellular pH is a key event in the mechanism of growth factor action.

Abstract: The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.

Abstract: Sensitization of the gill withdrawal reflex results from presynaptic facilitation at the excitatory synapses made by sensory neurons on gill motor neurons. Facilitation is accompanied by an increase in the duration of the action potential in sensory cells because of the depression of a K+ current. This results in an increasd influx of CA2+ and a greater release of transmitter from sensory neurons. There is evidence that serotonin is the facilitating transmitter and that the depression of the K+ current by serotonin mediated by cAMP-dependent protein phosphorylation. To test further the role of the cAMP-dependent protein kinase and of protein phosphorylation in sensitization, we have attempted to prevent or reverse the development of the electrophysiological correlates that accompany sensitization. We have pressure-injected sensory neurons with a specific and a stable protein inhibitor of the cAMP-dependent protein kinase both before and after the application of serotonin or the activation of the facilitator neurons. The increase in spike broadening that accompanies facilitation was prevented or diminished by injection of the inhibitor. Moreover, injection of the inhibitor could reverse fully the developed spike broadening produced by prior application of serotonin. These observations strenthen the evidence for the involvement of protein phosphorylation in presynaptic facilitation. Phosphorylation of the substrate protein evidently is quite labile and does not persist after the kinase is inhibited. Thus, the time course of short term sensitization appears to be determined by an active kinase. We think that it is likely that the mechanism for maintaining the kinase in an active form resides in the slow decay of the cAMP produced by the action of serotonin or the facilitator neurons on the sensory cells.

Abstract: The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.

Abstract: A431 cell membranes phosphorylate a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gly) in which residues 2--12 correspond to the sequence of the reported site of tyrosine phosphorylation in pp60src. Epidermal growth factor stimulates the phosphorylation of this peptide 2-fold over basal levels in a dose-dependent fashion. Phosphorylation is linear for approximately 3 min at 30 degrees C and occurs on the tyrosine residue. Kinetic analysis of the phosphorylation reaction indicates that epidermal growth factor increases the average Vmax from 3.8 to 7.5 nmol/min per mg and slightly decreases the average Km from 0.53 mM to 0.28 mM. A number of other peptides analogous to this tridecapeptide are also phosphorylated by A431 membranes. The data suggest that peptides with sequences similar to the site of tyrosine phosphorylation in pp60src are preferred substrates for the kinase in these membranes. Thus, the epidermal growth factor-stimulated protein kinase has the potential to interact with and phosphorylate pp60src. However, the A431 membranes also phosphorylate a tyrosine-containing peptide of totally unrelated sequence, suggesting that the kinase possesses a broad specificity for peptide phosphorylation that may not reflect its specificity with protein substrates.

Abstract: Depolarizing voltage steps induce inward and outward currents in voltage-clamped, internally perfused neurons from the snail Helix roseneri. Addition of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) to the internal perfusing medium results in an increase in the net outward current, with no apparent effect on the inward current. Catalytic subunit inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) is without effect, indicating that the increase in net outward current results from protein phosphorylation rather than an unspecific effect of protein perfusion. Decreasing the external Ca2+ concentration from 10 to 1 mM eliminates the effect of catalytic subunit, suggesting that Ca2+ plays an important role in this response. This suggestion is supported by the fact that the stimulation by catalytic subunit can be mimicked by increasing the Ca2+ concentration in the internal perfusion medium and can be prevented by intracellular perfusion with 10 mM EGTA. The results are consistent with the hypothesis that cyclic AMP-dependent protein phosphorylation regulates the Ca2+-activated K+ conductance in these cells.

Abstract: Ca2-dependent protein phosphorylations activated by calmodulin or phospholipid were studied using selective inhibitors. Both protein phosphorylations were inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and its derivatives. Kinetic analysis indicated that the primary effect of these agents was mediated through a competitive inhibition of enzyme activation by interaction with calmodulin or phospholipid, and Ki values of W-7 for calmodulin-dependent phosphorylation and phospholipid-dependent protein kinase were 12 microM and 110 microM, respectively. The addition of Ca2+ inhibited the binding of [3H]W-7 to phosphatidylserine but not the binding to calmodulin. The potencies of naphthalenesulfonamide derivatives as derivatives as inhibitors of Ca2+, calmodulin-dependent protein kinase were dependent on the length of the alkyl chain (C2-C10) but not on Ca2+-activated, phospholipid-dependent protein kinase. These results suggest that naphthalenesulfonamide derivatives may be more selective inhibitors of Ca2+, calmodulin-dependent protein phosphorylation than is Ca2+-activated, phospholipid-dependent protein kinase and that the mechanism of interaction between W-7 and phosphatidylserine differs from the interaction between W-7 and calmodulin. These agents are useful tools for elucidating the physiological role of Ca2+-dependent protein phosphorylation.

Abstract: The localization of the phosphoprotein B-50 (molecular weight 48,000, isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an immunochemical detection method employing an anti-B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1–24 is discussed.

Abstract: Cyclic adenosine monophosphate (AMP) analogs or agents that increase intracellular cyclic AMP rapidly stimulate transcription of the prolactin gene in a line of cultured rat pituitary cells. This effect is correlated with the phosphorylation of a chromatin-associated basic protein designated BPR. These data are consistent with the postulate that increased intracellular cyclic AMP concentrations induce rapid transcriptional effects on specific genes in eukaryotes, mediated by direct or indirect phosphorylation of a specific chromatin-associated protein or proteins.

Abstract: A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor.

Abstract: There is extensive evidence that protein synthesis in reticulocyte lysates is inhibited during haem deficiency because of phosphorylation of initiation factor eIF-2 by a protein kinase (the haem-controlled represser, HCR)1–5. Other protein kinases, which are activated by low concentrations of double-stranded RNA, have also been identified in reticulocytes1,6 and in extracts from interferon-treated cells7–10, and these phosphorylate an identical site on eIF-2 11–12. Despite the extensive documentation of such enzymes, however, the precise mechanism of inhibition has remained unclear, mainly because phosphorylation fails to affect many partial reactions of polypeptide chain initiation13,14. We present here evidence that the exchange of GTP for GDP bound to eIF-2 is inhibited following phosphorylation of the factor. Such an effect may inactivate eIF-2 by impairing the ability to recycle between successive rounds of protein synthesis.

Abstract: Ribosomes isolated from Drosophila melanogaster tissue culture cells labeled in vivo with 32Pi contain a single, heavily phosphorylated, ribosomal protein. As much as 40% of this protein is phosphorylated in cells cultured at 25 degrees C. The molecular weight and other characteristics of this protein suggest possible homology with ribosomal protein S6. Following a shift-up to 37 degrees C, the protein is specifically and quantitatively dephosphorylated. The kinetics of this dephosphorylation are rapid with a half-time on the order of a few minutes. These kinetics closely parallel the heat shock-induced breakdown of the preexisting polysome population.

Abstract: 1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 mM EGTA, with or without further fractionation, showed that Ca2+-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca2+-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca2+-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7).

Abstract: Rat embryo fibroblasts transformed by Abelson murine leukemia virus (MuLV) produce and release a transforming growth factor (TGF). Production of this factor is correlated with a tyrosine-specific protein kinase that is functionally active and is associated with the major Abelson MuLV gene product, P120. Transformation-defective mutants of Abelson MuLV do not transform cells, do not have their virus coded transforming gene product phosphorylated in tyrosine, and do not induce TGF production. Abelson MuLV-induced TGF morphologically transforms cells in culture, competes with 125I-labeled epidermal growth factor (EGF) for binding to cell receptors, and induces phosphorylation of tyrosine acceptor sites in the 160,000-dalton EGF membrane receptor. After purification to homogeneity, Abelson virus-induced TGF migrates as a single polypeptide with an apparent size of 7400 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.