Exogenous Phosphorylation Research Articles

NiSe@Ni12P5 hierarchical nanorod arrays coupled on nickel-copper foam for highly efficient urea oxidation

Electrocatalytic urea oxidation reaction (UOR) is one of the most promising energy-saving alternatives to anodic oxygen evolution reaction (OER), and the development of efficient and stable catalysts for UOR is crucial. Therefore, in this paper, hierarchical structure NiSe@Ni12P5/NCF with Ni12P5 nanorod arrays coupled with NiSe nanospheres are synthesized on bare nickel-copper foam (NCF) by exogenous phosphorylation and subsequent electrodeposition methods. The catalytic performance of hierarchical structure NiSe@Ni12P5/NCF in urea-assisted electrolytic water reaction is studied in focus. The results show that the potential of NiSe@Ni12P5/NCF was only 1.267 V and 1.412 V vs RHE at current densities of 10 mA cm−2 and 100 mA cm−2, respectively, in addition to no significant activity decay after 140 h stability test. This is attributed to the fact that the introduction of NiSe regulated the interfacial charge distribution, creating an interfacial coupling effect that can greatly increase the original catalytic species, as well as the NiOOH species generated during the UOR process as true active sites, which ultimately improving the electrocatalytic UOR performance of NiSe@Ni12P5/NCF. This study provides a new idea for the preparation of hierarchical selenide - phosphides for electrochemical applications.

Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast.

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flag-tagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.

Site-Specific 32 P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for several purposes. Because many protein kinases, each preferring a unique consensus sequence, are well characterized, the essential structure and function of the target protein can be effectively preserved through judicious selection and design of the phosphate incorporation site. After phosphorylation, these proteins are often indistinguishable from the parent molecules in assays of functional or biological activity. This convenient approach permits incorporation of 32P, 33P, 35S, or nonradioactive 31P, and is rapid, efficient, and safe. Most importantly 32P labeling of monoclonal antibodies or other therapeutic protein candidates has several significant advantages over radioiodination or chemical conjugation of heavy metal isotopes.

Diabetic alterations in cardiac sarcoplasmic reticulum Ca 2+-ATPase and phospholamban protein expression

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca 2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca 2+-ATPase and phospholamban (the inhibitor of SR Ca 2+-ATPase). The maximal Ca 2+ uptake and the affinity of Ca 2+-ATPase for Ca 2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca 2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca 2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca 2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca 2+-ATPase level would contribute to decreased rates of SR Ca 2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.

430. Transductional mechanisms and gene expression in melancholia

We have shown that melancholic depressives have a reduction in the exogenous phosphorylation activity of protein kinase A (PKA) relative to normal volunteers using cultured skin fibroblasts. In this study we contrasted melancholics and normals with regard to three factors: (1) The binding of [H] cyclic AMP to PKA; (2) The phosphorylation of endogenous CREB; and (3) The alteration of the expression of a CREB/CRE-dependent transfected luciferase reporter gene. Methods: Fibroblasts were cultured from drug-free subjects with DSM-IV major depression (melancholic type) and normal volunteers. [H] cyclic AMP binding (7 nM) was determined in the 48,000 3 g supernatant fraction. Cultures were incubated with 1 mM isoproterenol for 10 minutes after which CREB/CREB-P were assayed by subjecting nuclear lysates to gel electrophoresis followed by immunoblotting using polyclonal anti-CREB/CREB-P. Fibroblasts were also stably transfected with the luciferase reporter plasmid pAD neo2-C 12-BGL to evaluate any alteration in cAMP-PKA-CREB-dependent gene expression. Results: [H] cyclic AMP binding was significantly reduced in melancholics (p , 0.02): 902 6 73 vs. 631 6 51 fmol/mg protein. Isoproterenol-stimulated CREB-P and luciferase gene expression are also reduced in fibroblasts from melancholics. Both of these effects were blocked by the beta-2 antagonist ICI 118551. Conclusions: The previous finding of an abnormality of PKA activity in melancholia are now extended to suggest a reduction in CREB-P formation and an alteration in the expression of a specific CREB-CREdependent genes. We also will present newer data regarding differential expression of endogenous genes by differential display.

Topographic regulation of cytoskeletal protein phosphorylation by multimeric complexes in the squid giant fiber system.

In mammalian and squid nervous systems, the phosphorylation of neurofilament proteins (NFs) seems to be topographically regulated. Although NFs and relevant kinases are synthesized in cell bodies, phosphorylation of NFs, particularly in the lys-ser-pro (KSP) repeats in NF-M and NF-H tail domains, seem to be restricted to axons. To explore the factors regulating the cellular compartmentalization of NF phosphorylation, we separated cell bodies (GFL) from axons in the squid stellate ganglion and compared the kinase activity in the respective lysates. Although total kinase activity was similar in each lysate, the profile of endogenous phosphorylated substrates was strikingly different. Neurofilament protein 220 (NF220), high-molecular-weight NF protein (HMW), and tubulin were the principal phosphorylated substrates in axoplasm, while tubulin was the principal GFL phosphorylated substrate, in addition to highly phosphorylated low-molecular-weight proteins. Western blot analysis showed that whereas both lysates contained similar kinases and cytoskeletal proteins, phosphorylated NF220 and HMW were completely absent from the GFL lysate. These differences were highlighted by P13(suc1) affinity chromatography, which revealed in axoplasm an active multimeric phosphorylation complex(es), enriched in cytoskeletal proteins and kinases; the equivalent P13 GFL complex exhibited six to 20 times less endogenous and exogenous phosphorylation activity, respectively, contained fewer cytoskeletal proteins and kinases, and expressed a qualitatively different cdc2-like kinase epitope, 34 kDa rather than 49 kDa. Cell bodies and axons share a similar repertoire of molecular consitutents; however, the data suggest that the cytoskeletal/kinase phosphorylation complexes extracted from each cellular compartment by P13 are fundamentally different.

Novel endogenous protein kinase C substrate proteins, neutrinins, in brain synaptic membranes of maturing rat.

A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC19-31 or Ca(2+)-chelating reagents and again stimulated by the PKC activator phorbol 12, 13-dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane-associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.

Synthetic Tyr-phospho and non-hydrolyzable phosphonopeptides as PTKs and TC-PTP inhibitors.

Tyrosine-specific protein kinases and phosphatases are important signal transducing enzymes in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. In order to develop agents which inhibits the function of these two classes of enzymes by interfering with the binding of their substrates, we synthesized analogs derived from the peptide EDNEYTA. This sequence reproduces the main autophosphorylation site of Src tyrosine kinases. In this work we report the synthesis, by classical solution methods, of the phosphotyrosyl peptide EDNEYpTA as well as of three analogs in which the phosphotyrosine is replaced by a phosphinotyrosine and by two unnatural, non-hydrolyzable amino acids 4-phosphonomethyl-L-phenylalanine and 4-phosphono-L-phenylalanine. The Src peptide and its derivatives were tested as inhibitors of three non-receptor tyrosine kinases (Lyn, belonging to the Src family, CSK and PTK-IIB) and a non-receptor protein tyrosine phosphatase obtained from human T-cell (TC-PTP). The biomimetic analogues, which do not significantly affect the activity of CSK, PTK-IIB and TC-PTP, act as efficient inhibitors on Lyn, influencing both the exogenous phosphorylation and, especially, its autophosphorylation. In particular, the Pphe derivative may provide a basis for the design of a class of inhibitors specific for Lyn and possibly Src tyrosine kinases, capable of being used in vivo and in vitro conditions.

Determination of v-Mos-Catalyzed Phosphorylation Sites and Autophosphorylation Sites on MAP Kinase Kinase by ESI/MS

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.

Abnormal cAMP-induced phosphorylation of rap 1 protein in grey platelet syndrome platelets.

We previously demonstrated abnormal Ca2+ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca2+ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca2+ ATPases as those recently described in normal platelets; the 100 kD SERCA2-b isoform (Sarco/Endoplasmic Reticulum Ca2+ATPase) and a new 97 kD SERCA isoform. The two Ca2+ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca2+ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 micrograms/ml) of the catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level. It is concluded that the abnormal Ca2+ transport found in grey platelet syndrome platelets is not due to the abnormal expression of the Ca2+ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.

Effects of exercise training on the relationship between insulin binding and insulin-stimulated tyrosine kinase activity in rat skeletal muscle

The effect of exercise training on insulin binding and insulin receptor tyrosine kinase activity was studied using detergent solubilized wheat germ agglutinin (WGA)-agarose purified receptor preparations from rat biceps femoris (BF) and tensor fascia lata (TFL) muscles. Insulin receptor activity, as assessed by A14 [ 125I] insulin binding, was significantly elevated in BF of exercise-trained rats when compared with similar preparations from a sedentary control group. This increase in binding activity was due to change in B max not K D. In contrast, neither the B max nor the K D of insulin binding to TFL changed with exercise training. The structure of insulin receptors isolated from BF or TFL was unaltered by exercise training as determined by affinity labeling (α-subunit, molecular weight (mol wt) ∼131 kilodaltons [kDa]) and electrophoretic mobility of the α- and β-subunit. Furthermore, basal tyrosine kinase activity was not affected by exercise training in extracts from either BF or TFL. However, the insulin dependent increase in maximal tyrosine kinase activity (V max) of the BF, but not TFL, was enhanced by exercise training. Specifically, insulin stimulated phosphorylation of both the β-subunit of the insulin receptor (auto phosphorylation) and of a synthetic peptide (exogenous phosphorylation) were increased over control values in BF from exercise-trained rats, whereas both measurements of tyrosine kinase activity of TFL from the two experimental groups were similar. In contrast, both insulin-stimulated autophosphorylation and tyrosine kinase activity were significantly decreased in BF of exercise-trained rats when normalized to insulin binding activity. This disassociation was only seen in BF from exercise-trained rats, and was not true of TFL. These data indicate that excercise training can lead to increases in insulin receptor number and tyrosine kinase activity, as well as modifying the relationship between these two variables. The changes noted are not observed in all exercising muscles, and their development seems to depend upon the fiber composition. These results emphasize the complex relationship that exists in the regulation of insulin action at the level of its receptor.

Further enzymatic characteristics of a thylakoid protein kinase.

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.

Glucose-induced phospholipid-dependent protein phosphorylation in neonatal rat islets

The participation of calcium-activated, phospholipid-dependent protein kinase in the phosphorylation of endogenous islet proteins following the exposure of cultured, neonatal pancreatic islets to stimulatory glucose concentrations was investigated by two techniques. In the first technique, islets were prelabeled with 32 P i. The major endogenous substrates for glucose-induced phosphorylation had apparent molecular masses of 130,100 ± 1010, 100,000 ± 700, 80,400 ± 890, 58,100 ± 1200, 39,800 ± 700, and 29,400 ± 700 Da. In the presence of 12- O-tetradecanoylphorbol 13-acetate (2 μ m), an activator of calcium-activated phospholipid-dependent kinase, there was enhanced phosphorylation of proteins of 80,000, 40,000, and 29,000 Da. In the second technique, exogenous phosphorylation by [γ- 32P]ATP of proteins in a postnuclear particulate fraction was studied in the presence and absence of cofactors for Ca 2+-activated, phospholipid-dependent protein kinase (Ca 2+, phosphatidylserine, and unsaturated diolein). These studies were performed in islets preexposed to low (1.7 m m) or high (16.7 m m) glucose concentration prior to preparation of the postnuclear particulate fraction. Following exposure of islets to low glucose concentration, three substrates (apparent molecular masses 40,500 ± 600, 57,100 ± 700, and 79,400 ± 600 Da) in the postnuclear particulate fraction exhibited enhanced phosphorylation in the presence of calcium ions, phosphatidylserine, and unsaturated diolein. In preparations of islets preexposed to 16.7 m m glucose, the phosphorylation of the protein of molecular mass about 40,000 Da was significantly reduced, indicating prior phosphorylation of the acceptor sites on this substrate in response to glucose exposure. It is concluded that stimulation of neonatal cultured islets by glucose induces the acute changes in calcium ion, phospholipid, and diacylglycerol concentration required to activate the calcium-activated phospholipid-dependent protein kinase and that the islet postnuclear particulate fraction contains at least one specific substrate for this kinase.

Polymyxin B causes coordinate inhibition of phorbol ester-induced C-kinase activity and proliferation of B lymphocytes

Lymphocytes were found to be rich in phospholipid Ca 2+ -dependent (C-kinase) activity. Addition of polymyxin B (PMB) to in vitro assays of endogenous and exogenous phosphorylation resulted in profound inhibition of C-kinase activity. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) directly activated C-kinase, leading to increased phosphorylation of the same substrates. TPA also stimulated proliferation of B cells as assessed by 3H-thymidine uptake, and PMB strongly inhibited this effect. This coordinate inhibition of TPA-induced phosphorylation and mitogenesis indicates that PMB is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.

Erythrocyte membrane phosphorylation in aging rats.

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein profile and autophosphorylation of plasma membranes of red cells from 3-, 9-, 18-, and 30-month-old rats was examined. No major changes in SDS-PAGE protein profiles were noticeable. However, endogenous (auto) and exogenous phosphorylation in the presence of (gamma-32P)ATP showed an age-dependent increase in 32P uptake. pH optimum was between 8.0 and 9.0 for all age groups. Cyclic AMP was without effect on either endogenous or exogenous phosphorylation. Further analysis of the autophosphorylation reaction by quantification of autoradiograms obtained from SDS-PAGE gels displayed an age-dependent increase in phosphorylation of bands 2, 3, 4.1, and 4.5 [nomenclature of Fairbanks et al. Biochemistry 10:2606, 1971] and of several other minor bands. The data imply that alteration in red cell membranes may occur during aging of rats causing an enhancement in endogenous kinase activity and possibly in the number of phosphate acceptor sites as well.

Cyclic AMP-stimulated protein kinase activity in rabbit peripheral myelin.

Cyclic AMP-sensitive protein kinase activity has been found in suspensions of purified rabbit peripheral myelin. The enzyme phosphorylated the P0, "Y", X, P1, and P2 myelin proteins. Kinase activity, which was maximal at physiological pH, 2.5 mM Mg2+, and 2 microM cAMP, was stimulated three-fold over basal levels by cyclic AMP. Addition of calcium or EGTA had no effect on the enzyme activity in the presence or absence of cyclic AMP. Cyclic GMP also did not stimulate endogenous or exogenous protein phosphorylation. Theophylline, an inhibitor of 3',5'-cyclic nucleotide phosphodiesterase activity, increased protein kinase activity in the presence of cyclic AMP. These data show that PNS myelin proteins can be phosphorylated in situ by a protein kinase system whose activity is stimulated selectively by cyclic AMP.

Effects of corticotropin-(1-24)-tetracosapeptide on polyphosphoinositide metabolism and protein phosphorylation in rabbit iris subcellular fractions.

Effects of the neuropeptide corticotropin-(1-24)-tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 X g supernatant fraction [30-50% (NH4)2SO4 precipitate; ASP 30-50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The 32P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [gamma-32P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP 30-50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30-60 s), and it was dependent on the presence of Mg2+ in the incubation medium; in general it was inhibited by high concentrations (greater than 0.2 mM) of Ca2+. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50-100 microM of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 microM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP 30-50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation by cyclic AMP in vitro of liver plasma membrane (Na+--K+)-ATPase and protein kinases.

Abstract The effect of cyclic AMP on (Na+—K+)-ATPase and protein kinase activities of isolated liver plasma membrane has been investigated. Theophylline and caffeine, several cyclic nucleotides (including cyclic AMP, dibutyryl cyclic AMP, cyclic GMP and cyclic UMP), and sodium butyrate reduced the activity of (Na+—K+)-ATPase. The most powerful inhibitor was dibutyryl cyclic AMP; however, its action is probably due to the combined effects of cyclic AMP and butyrate. The blocking agent propranolol prevented the action of both epinephrine and cyclic AMP. Liver plasma membranes contain protein kinase(s), which catalyze the transfer of terminal phosphate of ATP into protein of plasma membrane itself (endogenous phosphorylation) as well as into added proteins (exogenous phosphorylation). Endogenous phosphorylation was inhibited by cyclic AMP, dibutyryl cyclic AMP and cyclic GMP. On the other hand, exogenous phosphorylation was stimulated by cyclic AMP. The specific activity of the membrane enzyme catalyzing the latter reaction was found to be higher than that of the enzyme present in the 100 000 × g supernatant of rat liver. The parallel effect of cyclic AMP on (Na+—K+)-ATPase and on the phosphorylation of membrane protein, and the finding that the latter reaction involves the formation of acylphosphate bonds, may be indicative of a possible role of endogenous phosphorylation in the regulation of (Na+—K+)-ATPase activity.