Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5′-O-(3-thiotriphosphate)[³⁵S] results in ³⁵S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or ³⁵S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4′diisothiocyanatostilbene-2,2′disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.     

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.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Regulation of protein synthesis in rabbit reticulocyte lysates. Thiophosphorylation of initiation factor eIF-2 by heme-regulated protein kinase

The heme-regulated protein kinase, which specifically phosphorylates the 38-kDa subunit of initiation factor eIF-2, can utilize adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) as a substrate. The rate of thiophosphorylation is 5-6-times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF-2 are resistant to dephosphorylation catalyzed by eIF-2 phosphoprotein phosphatase. The thiophosphorylated eIF-2 is less effective in promoting protein synthesis in hemin-deficient lysates under physiological conditions. In addition, ATP[gamma S] could also be utilized by the self-phosphorylation activity intrinsically associated with HRI.     

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.     

Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases

(H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

Functional shift from muscarinic to nicotinic cholinergic receptors involved in inositol trisphosphate and cyclic GMP accumulation during the primary culture of adrenal chromaffin cells.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.     

Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.     

Translocation of pleckstrin requires its phosphorylation and newly formed ligands

Pleckstrin is the major substrate of protein kinase C (PKC) in platelets. We sought to determine whether pleckstrin phosphorylation is sufficient to target the soluble protein to binding sites. Permeabilization of platelets by streptolysin O (SLO) was used to separate bound and soluble pleckstrin. Platelets were incubated with phorbol 12-myristate 13-acetate (PMA) and/or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the presence of [gamma-(32)P]ATP and SLO. PMA stimulated pleckstrin phosphorylation, but this pleckstrin diffused from permeabilized platelets. Addition of GTP[S] with PMA caused up to 40-50% of pleckstrin to be retained within platelets and enhanced secretion of platelet 5-hydroxytryptamine. PKC alpha pseudosubstrate peptide inhibited pleckstrin phosphorylation, the binding of pleckstrin and secretion. After extraction of permeabilized platelets containing bound pleckstrin with Triton X-100, the protein was solubilized. Thus, phosphorylated pleckstrin was retained in platelets only after activation of GTP-binding proteins that stimulate the formation of membrane-bound pleckstrin ligands. Translocation of pleckstrin may facilitate the associated secretion.     

Oligosaccharide signaling in plants. Specificity of oligouronide-enhanced plasma membrane protein phosphorylation

The in vitro phosphorylation by [gamma-32P]ATP of a 34-kDa plasma membrane-associated protein (pp34) from tomato and potato is strongly enhanced in the presence of alpha-1,4-D-polygalacturonic acid (PGA) fragments (Farmer, E. E., Pearce, G., and Ryan, C. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1539-1542) that activate the expression of defensive and developmental genes in plant tissues. [gamma-35S]ATP, but not [gamma-35S]GTP, has now been found to strongly label pp34 in the presence of the PGA fragments. PGA-enhanced phosphorylation of pp34 is at one or more threonine residue(s) and therefore is the product of a serine/threonine kinase. alpha-1,4-L-Polyguluronic acid (PGU) enhances thiophosphorylation of pp34, but is less effective than PGA. beta-1,4-D-Polymannuronic acid (PMA) is inactive. In vivo synthesis of proteinase inhibitors in tomato leaves in response to PGA, PGU, and PMA parallels enhancing activities in in vitro phosphorylation assays. The minimum oligogalacturonide lengths that enhance in vitro thiophosphorylation of pp34 are about 14-15 residues, which are near the minimum sizes of uronides required to elicit a variety of localized defensive and developmental responses in plants. The lengths of biologically active galacturonic acid oligomers are of the same length that form strong intermolecular complexes in solution with Ca2+. Uronide-Ca2+ complexes are proposed to be the active molecular species that initiate the signal transduction pathways regulating uronide-responsive genes.     

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.     

Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.     

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

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