A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed.     

Abnormal behavior of protein kinase C in the human myeloma cell line, RPMI 8226

Protein kinase C activity of the human myeloma cell line, RPMI 8226, was studied after prepurification on DEAE-cellulose. The total protein kinase activity, eluted at 0.12 M NaCl, was 493 nmol/min/10(10) cells, but 38% was associated with membranes. The lipid dependence of cytosolic and membrane activities was only 52% and 21%, respectively. This activity increased with time, to as much as 200% for the membrane fraction after 7 days, whereas lipid dependence and the PDBu binding properties were lost. This modified activity was not due to the extinction of a copurifying endogenous inhibitor nor to classical PKC proteolysis. TPA-treatment of these cels is accompanied by a rapid, selective and complete loss of lipid-dependent activity of the cytosol, thus benefiting co-migrating lipid independent activity, with no membrane fraction recovery or PKM formation.     

Purification and characterization of a novel calcium-dependent protein kinase from soybean

A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A.C., Putnam-Evans, C.L., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and Blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 microM free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2(+)-ATP of 8 microM and a Vmax of 1.7 mumol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2(+)-GTP, in addition to Mg2(+)-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.     

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.     

Phosphate-limited oat. The plasma membrane and the tonoplast as major targets for phospholipid-to-glycolipid replacement and stimulation of phospholipases in the plasma membrane

We recently reported that cultivation of oat (Avena sativa L.) without phosphate resulted in plasma membrane phosphoglycerolipids being replaced to a large extent by digalactosyldiacylglycerol (DGDG) (Andersson, M. X., Stridh, M. H., Larsson, K. E., Liljenberg, C., and Sandelius, A. S. (2003) FEBS Lett. 537, 128-132). We report here that DGDG is not the only non-phosphorous-containing lipid that replaces phospholipids but that also the content of glucosylceramides and sterolglycosides increased in plasma membranes as a response to phosphate starvation. In addition, phosphate deficiency induced similar changes in lipid composition in the tonoplast. The phospholipid-to-glycolipid replacement apparently did not occur to any greater extent in endoplasmic reticulum, Golgi apparatus, or mitochondrial inner membranes. In contrast to the marked effects on lipid composition, the polypeptide patterns were largely similar between root plasma membranes from well-fertilized and phosphate-limited oat, although the latter condition induced at least four polypeptides, including a chaperone of the HSP80 or HSP90 family, a phosphate transporter, and a bacterial-type phosphoesterase. The latter polypeptide reacted with an antibody raised against a phosphate deficiency-induced phospholipase C from Arabidopsis thaliana (Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K., and Ohta, H. (2005) J. Biol. Chem. 280, 7469-7476). In plasma membranes from oat, however, a phospholipase D-type activity and a phosphatidic acid phosphatase were the dominant lipase activities induced by phosphate deficiency. Our results reflect a highly developed plasticity in the lipid composition of the plasma membrane and the tonoplast. In addition, phosphate deficiency-induced alterations in plasma membrane lipid composition may involve different sets of lipid-metabolizing enzymes in different plant tissues or species, at different stages of plant development and/or at different stages of stress adjustments.     

Hormonal regulation of a calcium-activated, phospholipid-dependent protein kinase in bovine adrenal cortex

The calcium-activated, phospholipid-dependent protein kinase (C kinase) and its proteolytic product (M kinase), originally discovered in central nervous tissue (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603-7610) were characterized in bovine adrenal cortex cytosol. An endogenous calcium-dependent protease able to generate M kinase from the isolated C kinase in vitro was also present in adrenocortical extracts. Bovine adrenocortical cells in suspension as well as in primary culture contain the C and the M kinase activities. Treatment of these cells by steroidogenic concentrations (nM to microM) of ACTH resulted in a time and dose-dependent increase of cytosolic C kinase activity, whereas no change in M kinase activity was detected. This apparent activation appears to result mostly from an intracellular shift of the membrane-associated C kinase to a soluble cytosolic form of the enzyme. These observations open the question of the possible implication of the calcium, phospholipid-dependent protein phosphorylation system in hormone-dependent cellular regulatory processes.     

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases)

Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

Structure-activity study of cytotoxicity and microtubule assembly in vitro by taxol and related taxanes

Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent M_r of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca²⁺ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Activation of Protein Kinase C by the Capsaicin Analogue Resiniferatoxin in Sensory Neurones

Abstract: Resiniferatoxin and capsaicin are sensory neurone-specific excitotoxins that operate a common cation channel in nociceptors. Resiniferatoxin is structurally similar to capsaicin and to phorbol esters. Specific [³H]-resiniferatoxin binding, which was detected in the membrane ( K _D value 1.8 ± 0.2 n M ) but not cytosolic fraction of rat dorsal root ganglia, could not be displaced by phorbol 12,13-dibutyrate. Conversely, resiniferatoxin did not displace [³H]phorbol 12,13-dibutyrate binding in either the cytosolic or membrane fraction. Resiniferatoxin and capsaicin both caused translocation of protein kinase C in dorsal root ganglion neurones (EC₅₀ value 18 ± 3 n M ). This translocation was greatly reduced but not abolished, in the absence of external Ca²⁺, suggesting that it was secondary to Ca²⁺ entry. Resiniferatoxin also caused direct activation of a Ca²⁺- and lipid-dependent kinase (or kinases) in the cytosolic fraction of dorsal root ganglia, at concentrations (100 n M to 10 µ M ) higher than required for displacement of [³H]resiniferatoxin binding or translocation of protein kinase C. Capsaicin (up to 10 µ M ) was unable to mimic this effect. These data imply that although resiniferatoxin-induced translocation of protein kinase C in dorsal root ganglion neurones was mainly indirect, it also caused direct activation of a protein kinase C-like kinase in these cells.     

EGF receptor affinity is regulated by intracellular calcium and protein kinase C

Phorbol esters specifically reduce the binding of epidermal growth factor to surface receptors in intact cells, but not when added directly to isolated membranes. We show that after treatment of intact cells with phorbol myristate acetate, 125I-EGF binding is reduced in membranes prepared subsequently. High-affinity binding of 125I-EGF is modulated by an intracellular calcium-dependent regulatory process. Preventing calcium entry with EGTA or enhancing intracellular calcium with A23187 in intact cells modulates EGF receptor affinity in membranes isolated subsequently. Also, EGTA attenuates the usual inhibition of EGF binding caused by phorbol esters. Membrane preparations do not respond to phorbol ester treatment because the calcium- and phospholipid-dependent protein kinase C is removed or inactivated during membrane isolation. Reconstitution of unresponsive membranes with purified C kinase alters phosphorylation of the EGF receptor and restores the inhibitory effect of phorbol esters on 125I-EGF binding previously observed only in intact cells. Thus, activation of the Ca++-dependent enzyme, C kinase, modulates EGF receptor affinity, possibly via altered receptor phosphorylation.     

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.     

The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane

Factor XI binds to activated platelets where it is efficiently activated by thrombin. The factor XI receptor is the platelet membrane glycoprotein (GP) Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), a significant fraction of which exists within lipid rafts on stimulated platelets (Shrimpton, C. N., Borthakur, G., Larrucea, S., Cruz, M. A., Dong, J. F., and Lopez, J. A. (2002) J. Exp. Med. 196, 1057-1066). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids implicated in localizing membrane ligands and in cellular signaling. We now show that factor XI was localized to lipid rafts in activated platelets ( approximately 8% of total bound) but not in resting platelets. Optimal binding of factor XI to membrane rafts required prothrombin (and Ca2+) or high molecular weight kininogen (and Zn2+), which are required for factor XI binding to platelets. An antibody to GPIb (SZ-2) that disrupts factor XI binding to the GPIb-IX-V complex also disrupted factor XI-raft association. The isolated recombinant Apple 3 domain of factor XI, which mediates factor XI binding to platelets, also completely displaces factor XI from membrane rafts. To investigate the physiological relevance of the factor XI-raft association, the structural integrity of lipid rafts was disrupted by cholesterol depletion utilizing methyl-beta-cyclodextrin. Cholesterol depletion completely prevented FXI binding to lipid rafts, and initial rates of factor XI activation by thrombin on activated platelets were inhibited >85%. We conclude that factor XI is localized to GPIb in membrane rafts and that this association is important for promoting the activation of factor XI by thrombin on the platelet surface.     

Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.