Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.     

In the absence of antibody IgGsorb precipitates human placenta phosphotyrosine-containing proteins

Lectin-purified human placenta plasma membrane proteins were phosphorylated in vitro. Mixing the reaction mixture with IgGsorb and incubation of the resultant pellet with p-nitrophenyl phosphate demonstrated the presence of phosphorylated-insulin receptor beta-subunit and a phosphorylated-180 kDa protein in acrylamide gel electrophoresis. The same two proteins were detected in the electrophoretic analyses of anti-phosphotyrosine immunoprecipitated phosphorylation reaction mixtures. In the absence of antibody, the amount of phosphorprotein in the IgGsorb pellet was dependent on the amount of IgGsorb added. IgGsorb did not precipitate 125I-labeled lectin-purified human placenta protein. Further, 10 mM O-phosphotyrosine completely inhibited the precipitation of phosphorylated human placenta proteins. These data suggest that IgGsorb specifically bound and precipitated phosphotyrosine-containing proteins in soluble human placenta plasma membranes.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Characterization of dual specificity protein kinase from maize seedlings

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Phosphorylation of endogenous and exogenous peptides by rat heart sarcolemma

Rat heart plasma membranes contain a calcium-dependent protein kinase which phosphorylates endogenous protein substrates as well as added histones. The major endogenous protein phosphorylated is of 17 kDa on SDS-polyacrylamide gel electrophoresis. Proteins of 85 kDa and 60 kDa were also phosphorylated. Treatment of a rat heart homogenate with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased the recovery of kinase activity in the sarcolemmal membranes by up to 10-fold. The activity in such membranes was no longer calcium dependent. Although several histones were effective substrates for the enzyme, myosin light chain and phosvitin were not phosphorylated. These membranes contain a very active ATP hydrolysing activity which necessitated very brief incubation times to avoid loss of substrate. The membranes also contain cyclic AMP dependent protein kinase activity which is not active unless cyclic AMP is added to the incubations. The calcium dependent endogenous kinase, which is not inhibited by the heat stable inhibitor protein of cyclic AMP-dependent kinase, or by trifluoperazine, has several properties in common with protein kinase C. Preincubation of the sarcolemmal membranes with a high concentration of insulin caused inhibition of the phosphorylation of the endogenous 17 kDa and 85 kDa bands. There was no effect on the phosphorylation of the 60 kDa peptide. This effect of insulin was specific for the hormone and required preincubation of the hormone with the membranes for 20 min.     

Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane.

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg²⁺ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Adenosine 3′,5′-monophosphate-dependent phosphorylation of a 6000 and A 22000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22 000 and 24 000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640–2647). The other protein that the ³²P label incorporates into has a molecular weight of approximately 6000. Like the 22 000 dalton protein, the 6000 dalton protein has characteristic of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the ³²P label is incorporated more rapidly into the 22 000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22 000 and the 6000 dalton protein are still phosphorylated but only to about a quarter of the activity found when cyclic AMP and protein kinase are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22 000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins.Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation, the phosphorylated 22 000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22 000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands

Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).     

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.     

Characteristics of thyroid protein kinase C. Different Ca2 requirement for the phosphorylation of endogenous proteins and of H1 histone

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP-dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities. Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (approximately 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I. The one-dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22-25 kDa and 32-36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins. A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc-specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one-dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins. Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two-dimensional PAGE into several distinct spots with molecular masses of 32-35 kDa and pI range of 5-7.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.