Cell surface expression of DBA binding sites in LLC-PK1 cells increases at confluence and is enhanced by PKA activation

Post-confluent populations of LLC-PK₁ cells express many glycoproteins at the surface, including glycoproteins which bind the lectin Dolichos biflorus agglutinin (DBA). DBA-binding glycoproteins are localized preferentially to the apical cell surface and exhibit molecular weights ranging from less than 30 kDa to greater than 200 kDa. Subconfluent cell populations exhibit little surface DBA binding. Upon attaining confluence DBA binding capacity increases progressively over several days. This correlates roughly with increasing activities of several differentiated apical membrane functions. Expression of DBA binding sites at the surface occurs on a cell-by-cell basis. An increasing level of surface DBA binding in the cell population corresponds to an increasing proportion of the cell population expressing binding sites. Activation of cAMP-dependent protein kinase in post-confluent cell populations, and to a lesser extent in subconfluent populations, increases surface DBA binding sites by a protein synthesis-dependent mechanism after a lag period of at least 24 h. This results from increases in both the proportion of cells expressing surface binding sites and the level of binding sites on individual cells. © 1993 Wiley-Liss, Inc.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

Organ-specific change inDolichos biflorus lectin binding by myocardial endothelial cells during in vitro cultivation

Summary Endothelial cells of the NMRI mouse strain express a cell surface glycoprotein recognized by the lectinDolichos biflorus agglutinin (DBA). This study documents a marked organ-specific increase in DBA-specific lectin binding of myocardium-derived endothelial cells (MEC) of the NMRI/GSF mouse during in vitro cultivation. An up to 20-fold increase in DBA binding sites is observed in long-term culture, an increase not found in other NMRI-derived endothelial cell lines (e.g., brain, aorta). The increase appears restricted to DBA in that binding with other lectins (PNA, WGA) was unaltered. NMRI MEC cultures maintain typical endothelial cell attributes such as cobblestone morphology on confluence, expression of endothelial cell-specific surface markers, and production of angiotensin-converting enzyme. Cultures routinely become aneuploid within 4 passages, several passages before upregulation of the DBA binding site(s). Myocardial endothelial cells sorted to obtain DBA^hi and DBA^lo cell populations generally maintained their sorted phenotype for 3 to 4 passages. Limiting dilution cloning resulted in clones varying in DBA expression. Clones for DBA^hi expression maintained their DBA affinity for at least 10 passages (>30 doublings), whereas DBA^lo clones gave rise to varying numbers of DBA^hi cells within 2 to 4 passages. We hypothesize that the change in DBA affinity accompanies in vitro aging, that the change is independent of alterations in karyotype, and that the increase in DBA affinity may reflect a change in one or more other endothelial cell properties. Additional studies will be necessary to determine whether the in vitro changes are correlated with specific functional alterations and whether they accurately reflect progressive changes of MEC in vivo.     

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.     

Increased level of cAMP-dependent protein kinase in aging human lung fibroblasts

Regulation of the expression of cAMP-dependent protein kinase in cellular aging was studied using the IMR-90 diploid human lung fibroblasts. The level of cAMP-dependent protein kinase present in cell extracts was monitored by 1) photoactivated incorporation of 8-N3-[32P]cAMP into the 47,000- and 54,000-dalton regulatory subunits of the type I and type II cAMP-dependent protein kinases, respectively; 2) cAMP-dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and 3) fractionation and analysis of the type I and type II cAMP-dependent protein kinase by DEAE-Sephacel column chromatography. Our results showed an approximately two- to threefold increase in the level of the type I cAMP-dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the "old" IMR-90 cells (population doubling greater than 48) as compared to that of the "young" cells (PDL 22-27). The timing of the increase in cAMP-dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP-dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3-L1) and myoblast (L-5, L-6, and C2C13) suggests that regulation of the levels of cAMP and cAMP-dependent protein kinase plays a significant role in the control of cell growth and differentiation.     

Nuclear translocation of cAMP-dependent protein kinase

A study was made of nuclear translocation of cAMP-dependent protein kinase and its subunits, as well as of the binding of these proteins to metaphase chromosomes. The CHO cell cultures were treated with ³H-labelled protein kinase and its subunits. The results indicate that cAMP-dependent protein kinase became translocated into the nucleus in a dissociated state and that the subunits have specific binding sites on chromatin. Transformation of normal mouse fibroblasts by virus SV40 interferes with the nuclear translocation of the regulatory subunit. The process is restored when the level of cAMP in the system is increased. Binding of the regulatory subunit to metaphase chromosomes of cells transformed by virus SV40 does not change. In the case of spontaneous cancer (KB cells) translocation of the regulatory subunit remains unaffected, whereas acceptance of the protein by the metaphase chromosomes is impeded. The results of this work suggest that compartmentalization of cAMP-dependent protein kinase—and particularly of its regulatory subunit—in the cell is highly significant for cellular processes. Disorders arising as a result of neoplastic transformation involve changes in nuclear translocation of the regulatory subunit and in its binding to the structural elements of the genome.     

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

Surface glycoproteins bearing alpha-GalNAc terminated chains accompany pyriform cell differentiation in lizards.

The present investigation demonstrates that in squamate reptiles, as already reported for Podarcis sicula (Andreuccetti et al., 2001), the differentiation of pyriform cells from small, stem follicle cells is characterized by the progressive appearance on the cell surface of glycoproteins bearing alpha-GalNAc terminated O-linked side chains. Using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that, during previtellogenesis, the pattern of distribution of DBA binding sites over the follicular epithelium dramatically changes. In fact, binding sites first appear in follicular epithelium at the time that small cells begin to differentiate; in such follicles, labeling is evident on the cell surfaces of small and intermediate cells. Later on, as the differentiation progresses, the binding sites also become evident on the cell surface of pyriform cells. Once differentiated, the pattern of the distribution of DBA binding sites over the follicular epithelium does not change. By contrast, during the phase of intermediate and pyriform cell regression, DBA binding sites gradually decrease, so that the monolayered follicular epithelium of vitellogenic follicles, constituted only by small cells, shows no binding sites for DBA. It is noteworthy that binding sites for DBA are present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina or among pyriform cells, and therefore not engaged in the differentiation into pyriform cells. This finding demonstrates that, in squamates, the pattern of distribution of alpha-N-GalNAc containing glycoproteins significantly changes during previtellogenesis, and that these modifications are probably related to the differentiation of small stem cells into highly specialized pyriforms.     

H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway

Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H₂O₂ (100 μM) for 1–60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H₂O₂. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H₂O₂ treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H₂O₂-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway. J. Cell. Physiol. 172:373–381, 1997. © 1997 Wiley-Liss, Inc.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

The active component of ginseng,ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

Multiple protein kinase activities in the dimorphic fungus Mucor rouxii. Comparison with a cyclic adenosine 3',5'-monophosphate binding protein.

Protein kinase and cyclic adenosine 3′,5′-monophosphate (cAMP) binding activities have been detected in cell extracts of the dimorphic fungus Mucor rouxii. The subcellular distribution of both activities indicates that most of the binding protein is in the high-speed supernatant (S₁₀₀), while about 70% of the total protein kinase activity remains in particulate fractions. S₁₀₀ preparations have been analyzed by diethylaminoethyl cellulose column chromatography. Binding activity can be resolved in two peaks (A and B) and protein kinase in three peaks (I, II, and III). Peaks I and II are casein dependent and insensitive to cAMP. Peak III utilizes histone as substrate and is activated (two- to fourfold) by cAMP. Theophylline strongly inhibits cAMP binding activity and mimics the effect of cAMP on cAMP-dependent protein kinase. The possible relationship between cAMP binding activity and cAMP-dependent protein kinase is suggested.     

Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit

BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.     

Binding of glutamate receptor delta2 to its scaffold protein, Delphilin, is regulated by PKA

The glutamate receptor delta2 (GluRdelta2) is selectively expressed in cerebellar Purkinje cells and plays an important role in motor learning, motor coordination, and long-term depression. Delphilin is identified as a GluRdelta2-interacting protein, selectively expressed in Purkinje cell-parallel fiber synapses, and specifically interacts with the GluRdelta2 C-terminus via its PDZ domain. Here, surface plasmon resonance analyses showed that Delphilin PDZ bound to GluRdelta2 C-terminal peptide (DPDRGTSI), but not to its phosphopeptides (DPDRGphosphoTSI and DPDRGTphosphoSI). We showed the incorporation of phosphate into threonine at -2 (-2T) and serine at -1 (-1S) of GluRdelta2 C-terminus by cAMP-dependent protein kinase (PKA) in vitro. In the experiments using heterologous expression system, Delphilin coimmunoprecipitated with GluRdelta2 was dramatically decreased under the condition with forskolin and isobutylmethylxanthine, which led to cAMP-dependent phosphorylation by PKA. Thus, phosphorylation of -2T and/or -1S of GluRdelta2 C-terminus by PKA may regulate the binding of GluRdelta2 to its scaffolding protein, Delphilin.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

Compartmentalized cAMP signalling in regulated exocytic processes in non-neuronal cells

Cyclic adenosine monophosphate (cAMP) is a central second messenger controlling a plethora of vital functions. Studies of cAMP dynamics in living cells have revealed markedly inhomogeneous concentrations of the second messenger in different compartments. Moreover, cAMP effectors such as cAMP-dependent protein kinase (PKA) and cAMP-activated GTP-exchange factors (Epacs) are tethered to specific cellular sites. Both the tailoring of cAMP concentrations, and the activities of cAMP-dependent signalling systems at specific cellular locations are prerequisites for most, if not all, cAMP-dependent processes. This review focuses on the role of compartmentalized cAMP signalling in exocytic processes in non-neuronal cells. Particularly, the insertion of aquaporin-2 into the plasma membrane of renal principal cells as an example for a cAMP-dependent exocytic process in a non-secretory cell type, renin secretion from juxtaglomerular cells as a cAMP-triggered exocytosis from an endocrine cell, insulin release from pancreatic beta-cells as a Ca2+-mediated and cAMP-potentiated exocytic processes in an endocrine cell, and cAMP- or Ca2+ -triggered H+ secretion from gastric parietal cells as an exocytic process in an exocrine cell are discussed. The selected examples of cAMP-regulated exocytic pathways are reviewed with regard to key proteins involved: adenylyl cyclases, phosphodiesterases, PKA, A kinase anchoring proteins (AKAPs) and Epacs.     

Mechanisms of vascular angiotensin II surface receptor regulation by epidermal growth factor

We investigated mechanisms by which epidermal growth factor (EGF) reduces angiotensin II (AngII) surface receptor density and stimulated actions in vascular smooth muscle cells (VSMC). EGF downregulated specific AngII radioligand binding in intact cultured rat aortic smooth muscle cells but not in cell membranes and also inhibited AngII-stimulated contractions of aortic segments. Inhibitors of cAMP-dependent kinases, PI-3 kinase, MAP kinase, cyclooxygenase, and calmodulin did not prevent EGF-mediated downregulation of AngII receptor binding, whereas the EGF receptor kinase inhibitor AG1478 did. Total cell AngII AT1a receptor protein content of EGF-treated and untreated cells, measured by immunoblotting, did not differ. Actinomycin D or cytochalasin D, which interacts with the cytoskeleton, but not the protein synthesis inhibitor cycloheximide, prevented EGF from downregulating AngII receptor binding. Consistently, EGF inhibited AngII-stimulated formation of inositol phosphates in the presence of cycloheximide but not in the presence of actinomycin D or cytochalasin D. In conclusion, EGF needs an intact signal transduction pathway to downregulate AngII surface receptor binding, possibly by altering cellular location of the receptors.     

ATP binding to a unique site in the type-1 S2- inositol 1,4,5-trisphosphate receptor defines susceptibility to phosphorylation by protein kinase A

The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly --> Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly --> Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation.