The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.     

Stimulation of synaptic membrane phosphorylation by a calcium and calmodulin independent heat stable cytosol factor

Synaptosomal cytosol contains a heat-stable factor that stimulates the endogenous phosphorylation of synaptic plasma membranes in a calcium-independent fashion. This factor can be distinguished from the calcium binding protein, calmodulin, and appears to affect the incorporation of phosphate into a specific synaptic membrane protein.     

Participation of calmodulin in the regulation of plasma membrane electric potential by intracellular calcium

The effect of Ca2+ on the electric potential and permeability of human erythrocyte membranes for K+ was investigated. An increase of K+ concentration in a medium containing a Ca-ionophore A 23187 causes hyperpolarization of the erythrocyte membrane (by 50-60 mV) due to a 70-fold increase of its permeability for K+ (K0.5 for Ca2+ in both cases is equal to 2-3 microM). Using calmodulin-deficient inside-out erythrocyte membrane vesicles, it was demonstrated that regulation of the transmembrane potentials by Ca2+ is mediated by its interaction with calmodulin (K0.5 for Ca2+ and calmodulin is equal to 2-3 microM and 100-150 nM, respectively). It was assumed that the Ca2+-calmodulin complex is involved in the functioning of the plasma membrane K+-channel.     

Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineruin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.     

Auxin-stimulated ATPase in membrane fractions from pumpkin hypocotyls (Cucurbita maxima L.)

Membrane fractions from Cucurbita maxima hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10^-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10^-6 M IAA. Without PAA, only a high concentration of 10^-4 M IAA was stimulatory, whereas 10^-6 M IAA had no apparent effect and 10^-8 M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10^-4 M PAA was activated only by 2,4-dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin.Abbrevations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 3,5-D 3,5-dichlorophenoxyacetic acid - IAA indol-3-acetic acid - PAA phenylacetic acid - MES (2-(N-morpholino))-ethanesulfonic acid - EDTA ethylenediamine tetraacetic acid     

Protein kinases, regulated by calcium and calmodulin: their structure, regulation and cellular functions

In this review we summarize and generalize the recent data on structure, regulation and physiological role of calcium/calmodulin-dependent protein kinases, or CaM kinases. CaM kinases are the family of structurally homologous enzymes, involved into a variety of Ca(2+)-induced cellular reactions through phosphorylation of target proteins. In recent years the quantity of these enzymes has exceeded twenty, mainly due to identification of new isozymic forms of already known CaM kinases. Using crystal structure analysis data, some researchers constructed molecular models of regulation and functioning of CaM kinases. Many reports of recent years are devoted to investigation of functions of CaM kinase isozymes and their role in various cellular processes.     

Identification of etioplast membranes in fractions from soybean hypocotyls

Three independent methods, one cytological and two biochemical, were used to estimate contributions of plastids and plastid fragments to various membrane fractions. In thin sections viewed by electron microscopy, KMnO₄ selectively enhanced the images of plastid membranes in situ as well as in isolated fractions. The amounts of plastid fragments in isolated membrane fractions were determined by electron microscopic morphometry of fractions fixed with KMnO₄ in conjunction with analysis of galactolipids and carotenoids. Monogalactosyl and digalactosyl diglyceride contents were directly correlated with the amount of plastid membranes in the fractions identified by electron microscope morphometry. Amounts of carotenoids also correlated with plastid membranes except at very low levels where estimates based on carotenoids exceeded those based on morphometry.     

Stimulation of tyrosine phosphorylation of rat brain membrane proteins by calmodulin

Calmodulin stimulates the alkali-resistant phosphorylation of peptides of 50 and 58-60 kDa in rat brain membrane. Phosphoamino acid analysis indicated a calmodulin stimulated increase of phosphotyrosine in these peptides. Calmodulin also stimulated the phosphorylation of these peptides at serine and threonine residues. This suggests the involvement of the calmodulin regulatory system in the effects of tyrosine protein kinases.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.     

Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K⁺-stimulated, vanadate-inhibited Mg²⁺ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.     

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

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The regulation of muscle phosphorylase kinase by calcium ions,calmodulin and troponin-C

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase $\text{in vivo}$ is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism for coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the δ - subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state.

Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45–49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.     

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.     

Myosin V: regulation by calcium, calmodulin, and the tail domain

Calcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell.     

Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low K(m) Ca2+-activated (PDE1C) and Ca2+-independent, rolipram-sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP-dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT-cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT-cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope-tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.