Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca²⁺ export mechanisms in rat brain synaptic membranes. Ca²⁺ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca²⁺ pump and by a low affinity/high capacity ATP-independent Na⁺-Ca²⁺ exchanger. Both Ca²⁺ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP>NAD>NADPH>NADH with IC₅₀ values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca²⁺ pump and with IC₅₀ values between 8 and 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP and ferricyanide inhibited the ATP-driven Ca²⁺ efflux mechanism but not the Na⁺-Ca²⁺ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca²⁺ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by eithercAMP-dependent protein kinase, Ca²⁺ calmodulin-dependent protein kinases, or protein kinase C.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - EGTA ethylenglycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - DCIP dichlorophenol-indophenol     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Regulation of Ca2+ influx in myocardial cells by beta adrenergic receptors,cyclic nucleotides,and phosphorylation

Calcium channels in the heart play a major role in cardiac function. These channels are modulated in a variety of ways, including protein phosphorylation. Cyclic AMP-mediated phosphorylation is the best understood phosphorylation mechanism which regulates calcium influx into cardiac cells. Binding of an agonist (e.g., a catecholamine) to the appropriate receptor stimulates production of cyclic AMP by adenylate cyclase. The cyclic AMP may subsequently bind to and activate a cyclic AMP-dependent protein kinase, which then can phosphorylate a number of substrates, including the calcium channel (or a closely-associated regulatory protein). This results in stimulation of the calcium channels, greater calcium influx, and increased contractility. The cyclic AMP system is not the only protein kinase system in the heart. Thus, the possibility exists that other protein kinases may also regulate the calcium channels and, hence, cardiac function. Recent evidence suggests that cyclic GMP-mediated phosphorylation may play a role opposite to cyclic AMP-mediated phosphorylation, i.e., inhibition of the calcium current rather than stimulation. Other recent evidence also suggests that a calcium/calmodulin-dependent protein kinase and calcium/phospholipid-dependent protein kinase (protein kinase C) may also regulate the myocardial calcium channels. Thus, protein phosphorylation may be a general mechanism whereby calcium channels and cardiac function are modulated under a variety of conditions.     

Introduction: Protein phosphorylation and signal transduction in bacteria

A single type of reversible protein-phosphorylating system, the ATP-dependent protein kinase/phosphatase system, is employed in signal transduction in eukaryotes. By contrast, recent work has revealed that three types of protein-phosphorylating systems mediate signal transduction in bacteria. These systems are (1) classical protein kinase/phosphatase systems, (2) sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological, structural, and mechanistic aspects of these three evolutionarily distinct systems are discussed in the papers of this written symposium. © 1993 Wiley-Liss, Inc.     

The plant Ca2+ -ATPase repertoire: biochemical features and physiological functions

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

Specific protein phosphorylation occurs in molluscan red blood cell ghosts in response to hypoosmotic stress

Summary The regulation of cellular volume upon exposure to hypoosmotic stress is accomplished by specific plasma membrane permeability changes that allow the efflux of certain intracellular solutes (osmolytes). The mechanism of this membrane permeability regulation is not understood; however, previous data implicate Ca²⁺ as an important component in the response. The regulation of protein phosphorylation is a pervasive aspect of celllular physiology that is often Ca²⁺ dependent. Therefore, we tested for osmotically induced protein phosphorylation as a possible mechanism by which Ca²⁺ may mediate osmotically dependent osmolyte efflux. We have found a rapid increase in³²P_i incorporation into two proteins in clam blood cell ghosts after exposure of the intact cells to a hypoosmotic medium. The osmotic component of the stress, not the ionic dilution, was the stimulus for the phosphorylations. The osmotically induced phosphorylation of both proteins was significantly inhibited when Ca²⁺ was omitted from the medium, or by the calmodulin antagonist. chlorpromazine. These results correlate temporally with cell volume recovery and osmolyte (specifically free amino acid) efflux. The two proteins that become phosphorylated in response to hypoosmotic stress may be involved in the regulation of plasma membrane permeability to organic solutes, and thus. contribute to hypoosmotic cell volume regulation.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5′-trihosphate (ATP) resulted in an inhibition of Ca²⁺ efflux. The ATP-induced Ca²⁺ accumulation as determined by the increase in phosphorylase a activity and the Ca²⁺ -sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis-[carboxymethyl] aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca²⁺, suggesting that the increased free Ca²⁺ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H-imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca²⁺ -calmodulin process may be involved in the initiation of cytotoxicity.     

Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle

The Ca²⁺ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na^– + K⁺ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (K_D) for the binding of [³H]nitrendipine and [³H]verapamil were 0.31 ± 0.04 nM and 4.1 ± 0.5 nM respectively, and displayed an average density of 0.55 ± 0.05 pmol/mg and 0.4 ± 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca²⁺2 antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [³H]PN200-110, [³H]verapamil and [³H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [³H]PN200-110 were enriched about 2500 fold as compared with the original homogenate and displayed a density of 28.5 ± 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170000, 140000, 130000, 100 000 and 53000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170 000 and 53000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170000, 100000, 53 000 and 28000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [³H]nitrendipine, [³H]PN200-110, [³H]verapamil and [³H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca²⁺ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.Abbreviations CHAPS 3-[-(3-cholamidopropyl) dimethylammonio]-propanesulphonate - SDS sodium dodecyl sulphate Scholar of the Ontario Heart and Stroke Foundation.     

Some Properties of Caldesmon and Calponin and the Participation of These Proteins in Regulation of Smooth Muscle Contraction and Cytoskeleton Formation

The interaction of caldesmon with different Ca²⁺-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca²⁺-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B" of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actinbinding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and a-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.     

Physiological role of mitochondrial Ca2+ transport

A model has been proposed in which mitochondrial Ca²⁺ ion transport serves to regulate mitochondrial matrix free Ca²⁺ ([Ca²⁺]_m), with the advantage to the animal that this allows the regulation of pyruvate dehydrogenase and the tricarboxylate cycle in response to energy demand. This article examines recent evidence for dehydrogenase activation and for increases in [Ca²⁺]_m in response to increased tissue energy demands, especially in cardiac myocytes and in heart. It critiques recent results on beat-to-beat variation in [Ca²⁺]_m in cardiac muscle and also briefly surveys the impact of mitochondrial Ca^2– transport on transient changes in cytosolic free Ca²⁺ in excitable tissues. Finally, it proposes that a failure to elevate [Ca²⁺]_m sufficiently in response to work load may underlie some cardiomyopathies of metabolic origin.     

Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains

Ca²⁺ and calmodulin (CaM), a key Ca²⁺ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca²⁺ and CaM in defense signaling, we used ³⁵S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound ³⁵S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca²⁺ whereas EGTA, a Ca²⁺ chelator, abolished binding, confirming that PICBP binds CaM in a Ca²⁺-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca²⁺-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca²⁺-dependent CaM-binding domains.