Abstract: Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.

Abstract: Ubiquitination is one of several ways in which cells modify their proteins. As for phosphorylation or acetylation, there are distinct enzymes for adding and removing Ub from the surfaces of protein substrates. The dynamic equilibration of Ub with cellular proteins is also typical of most posttranslational modifications. Ubiquitination differs, however, in that the added group is large compared to acetate or phosphate. Its size must provide great potential for recognition by other cellular proteins. Ub may be the cell's reversible cross-linking reagent, covalently bound to protein substrates at one end and noncovalently associated with various Ub binding proteins at the other. It is likely that one ubiquitin binding protein is a component of the 26S ATP-dependent protease. The presence of Ub on histones and on the lymphocyte homing receptor suggests that ubiquitination does not serve exclusively to mark proteins for degradation. There are probably various ubiquitin binding proteins since Ub appears to be a multifunctional protein that affects chromatin structure, intracellular proteolysis, cellular interactions, and the stress response. This abundant protein may serve as an intracellular barometer whose distribution among several pools regulates a variety of processes.

Abstract: A general chemical phosphorylation method based on P(III) chemistry has been developed. The system is demonstrated for the phosphorylation of oligonucleotides, directly after their synthesis on a solid support, and for the O-phosphorylation of serine, threonine, and tyrosine as well as for a serine-containing peptide.

Abstract: The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.

Abstract: The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis.

Abstract: We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.

Abstract: Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.

Abstract: An assay has been developed to assess the dynamics of cell surface glycoproteins, in which neuraminidase digestion of intact cells is used to determine the fate of cell surface molecules initially labelled via lactoperoxidase-catalyzed iodination. This approach has been used to demonstrate the constitutive endocytosis and recycling of the T3-T cell receptor (T CR) complex on the human T leukemic cell line HPB-MLT. Stable populations of both phosphorylated and nonphosphorylated forms of the T3 gamma peptide have been identified in these cells. Whereas the former are constitutively endocytosed, the latter appear to be excluded from this pathway. The results presented indicate that T3 gamma phosphorylation may control the endocytosis and recycling of the T3-TCR complex on this cell line.

Abstract: Insulin stimulates phosphorylation of a tyrosine residue(s) on a 15-kDa protein (p15), and the cytosolic phosphorylated protein (pp15) accumulates only when 3T3-L1 adipocytes are treated with phenylarsine oxide It has been shown previously that phenylarsine oxide, an agent that complexes vicinal dithiols, interrupts signal transmission from the insulin receptor to the glucose transport system Several lines of evidence presented here indicate the involvement of pp15 in insulin receptor-initiated signal transduction to the glucose transport system The reciprocal effects of phenylarsine oxide on the insulin-activated accumulation of pp15 and on insulin-stimulated hexose uptake are reversed by the vicinal dithiol 2,3-dimercaptopropanol but not by the monothiol 2-mercaptoethanol Thus, a cellular dithiol appears to function in the signal transmission pathway downstream from pp15 Like the insulin-activated autophosphorylation of the receptor's beta subunit (on tyrosine), activation of phosphorylation of p15 is specific, with insulin-like growth factors 1 and 2, epidermal growth factor, and platelet-derived growth factor being inactive Moreover, both processes exhibit identical insulin concentration dependence The temporal kinetic relationship of insulin-activated receptor beta-subunit phosphorylation, followed by the phosphorylation of p15 and then increased hexose uptake rate, is consistent with an intermediary signaling role for pp15 in insulin-stimulated glucose uptake

Abstract: Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [ 32 P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a γ-[ 32 P]ATP-containing reaction mixture.

Abstract: Expression of the middle-T antigen of polyomavirus is sufficient to induce transformation of fibroblasts in culture and tumour formation in whole animals1–4. Middle-T can form a complex with the cellular src gene product (p60c-src) and can be phosphorylated by p60c-src in vitro5,6. Studies using middle-T mutants have suggested that the association of middle-T with pc-src may be necessary but not sufficient for transformation7–9. Therefore, we addressed the possibility that middle-T could interact with other tyrosine protein kinases structurally related to p60c-src. Using antibody raised against a fusion protein between β-galactosidase and amino-terminal sequences of p90gag-yes from Y73 virus (anti-yes antibody)10, we have found that middle-T can associate with and be phosphorylated by the c-yes proto-oncogene product, a protein of relative molecular mass (Mr) 62,000 (62K). This raises the possibility that the middle-T–c-yes complex contributes to transformation by polyomavirus.

Abstract: The role of substrate in influencing the cofactor requirements of the phospholipid- and Ca2+-dependent protein kinase C (PKC) was investigated by using several substrates. All of the substrates tested, including histone, troponin I, myosin light chain, protamine, poly(arginine, serine) (PAS), poly(lysine, serine) (PLS), and myelin basic protein (MBP), were found to interact with and aggregate phospholipid vesicles as well as phosphatidylserine (PS)-Triton mixed micelles. Phosphorylation of these different substrates by PKC indicated the presence of three distinct substrate categories: substrates such as protamine requiring no cofactors; substrates such as PLS, PAS, and MBP requiring only the presence of phospholipid; and substrates such as histone, myosin light chain, and troponin I requiring the presence of Ca2+ and phospholipid. Diacylglycerol was a major cofactor only with category C substrates. These different requirements correlated with the interaction of the substrate with phospholipid and/or enzyme. The substrates in category A interacted strongly with and aggregated PKC in a binary mixture. In the absence of Ca2+, PKC bound to substrates of category B directly but not to substrates in category C. Thus, substrate-enzyme binding eliminated the Ca2+ requirement of phosphorylation, and aggregation of substrate-enzyme complex eliminated the phospholipid requirements as well. Substrate-phospholipid interaction and substrate phosphorylation were inhibited by increasing salt concentrations, but the amount needed depended upon the substrate. Loss of PKC activity appeared to coincide with loss of substrate-PS aggregation while dissociation of PKC from the membranes required much higher salt concentrations. Poly(L-lysine) and poly(L-arginine), two potent inhibitors of PKC, also showed substrate-dependent inhibition characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.

Abstract: Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 μM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masscs, Mr, of 240000, 68 000, 50 000, and 22 000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1–2 μM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 μM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50000 which was also phosphorylated in response to low concentrations (1–2 μM) of cGMP in platelet membranes. An additional protein (Mr 24000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.

Abstract: The expression of the tyrosine aminotransferase (TAT) gene of the rat was analyzed in primary hepatocytes. The TAT gene remains active in primary cultured cells at a level similar to that in liver cells. Expression can be induced by glucocorticoids and cAMP, glucocorticoids lead to a 8–10-fold increase in TAT mRNA level, cAMP to a 20–30-fold increase. The elevation of the TAT mRNA is preceeded by a rise in the relative rate of transcription of the gene. Surprisingly transcription of the albumin gene, which steadily declines with the age of the culture, can also strongly be stimulated by glucocorticoids in primary hepatocytes. cAMP antagonists, which act as competitive inhibitors of the cAMP-dependent protein kinase, prevent induction of transcription of the tyrosine aminotransferase gene by cAMP suggesting that the effect of cAMP on expression of the tyrosine aminotransferase gene is mediated by a cAMP-dependent protein kinase. The cAMP antagonist does not interfere with induction by glucocorticoids which suggests that phosphorylation of the glucocorticoid receptor by the cAMP-dependent protein kinase is not required for its function. We thus conclude that the two inducers affect transcription by independent mechanisms.

Abstract: K+ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25×10−13 cm2/s; outward currents saturated under a high driving force for K+ movement. Internal 0.1–5.0 mM Mg2+, 0.1 μM Ca2+ and 10 mM Na+ each depressed the flux of K+ ions moving outwards through open channels. Internal 0.1–5.0 mM Mg2+, 0.1–1.0 μM Ca2+ and 1–10 μM Ba2+ and Sr2+ blocked K+ channel activity in a dose-and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATPγS or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca2+ (>=1 μM) and Sr2+ (1 mM) markedly inactivated K+ ATP channels, millimolar Ba2+ or Mg2+ were less effective. This suggested that the run down of the channels was a Ca2+-dependent dephosphorylation of the K+ channel protein.

Abstract: Drug-resistant cell lines derived from the mouse macrophage-like cell line J7742 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons) Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance