Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line.

Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-1 times/min) to thrombin (0.001-0.003U/ml). Ca++ transients appears to be fused with higher thrombin doses. With extracellular Ca++ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca++]i. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Ca++ oscillation, suggesting an important role of Ca++ influx for Ca++ oscillation.     

Production of platelet-like particles by a human megakaryoblastic leukemia cell line (MEG-01)

The distribution of microtubules and platelet-specific glycoproteins (GPIIb/IIIa) in particles was probed by an immunofluorescence method using anti-tubulin and anti-GPIIb/IIIa antibodies to identify whether particles released from a human megakaryoblastic cell line (MEG-01) are platelets. The fluorescence image showing anti-tubulin staining of the particles revealed a characteristic ring structure observed in platelets. Anti-platelet GPIIb/IIIa antibody staining showed an image in which small patches or spots were seen throughout the particle with brighter staining at the periphery. No significant difference was observed between these particles and human blood platelets under immunofluorescent staining. These results show that MEG-01 cells released platelet-like particles.     

Protein kinase C of a human megakaryoblastic leukemic cell line (MEG-01). Analysis of subspecies and activation by diacylglycerol and free fatty acids

Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG-01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (beta I/beta II) and type III (alpha) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 x 10(-5) M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor-mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.     

Identification of heterotrimeric GTP-binding proteins in human megakaryoblastic leukemia cell line,MEG-01, and their alteration during cellular differentiation

Various heterotrimeric GTP-binding proteins (G proteins) are possible to have important functions in hematopoietic cells. However, there has been no information regarding their expression in magakaryoblasts and/or megakaryocytes. In the present study, protein contents of seven G protein α subunits (Gs α, Gi2 α, Gi3 α, Gz α, G11 α, Gq α and G12 α) and β subunit in a human megakaryoblastic leukemia cell line, MEG-01, were analyzed by immunoblotting. Immature MEG-01 cells expressed the α subunits of Gs, Gi2, Gi3, Gz, G11 and G12 at protein molecule level. During the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced differentiation process, the contents of Gi2 α and Gi3 α increased, whereas the protein levels of Gz α, Gs α, Gil a and G12 α were observed to hardly change, β Subunit was also observed to be present in immature MEG-01 cells and to increase continuously throughout the differentiation process. For the expression of Gi2 α and β subunits, chronic TPA-treatment was required although Rac2, a low Mr GTP-binding protein, was expressed abundantly by only 30 min-TPA-treatment followed by 3 day-culture.     

Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association

Immunological analysis using monoclonal antibodies against subspecies of protein kinase C revealed the predominant expression of the isozyme, type II, in human megakaryoblastic leukemic cells. We investigated the effects of phorbol diester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the Ca2+ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) on the immunocytochemical localization of protein kinase C in these cells. Indirect immunofluorescence techniques revealed the enzyme to be located in a diffuse cytosolic pattern, in the intact cells. When the cells were exposed to 100 nM TPA, the immunofluorescent staining was translocated from the cytoplasm to the plasma membrane. The translocation was protracted and staining on the membrane decreased in parallel with the Ca2+, phospholipid-dependent protein kinase activity. Treatment of the cells with 500 nM ionomycin caused an apparent translocation comparable with that seen with TPA, however, this translocation was transient and most of the cytosolic staining was within 60 min. We also found that 30 micrograms/ml OAG did not have significant effects on distribution of the staining, but rather acted synergistically on the translocation with the suboptimal concentration of 100 nM ionomycin. A similar synergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca2+ and diacylglycerol regulate membrane binding of the enzyme in vivo.     

Thrombin-induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in bovine pulmonary artery endothelial cells

Myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate that is targeted to the plasma membrane by an amino-terminal myristoyl group. In its nonphosphorylated form, MARCKS cross-links F-actin and binds calmodulin (CaM) reciprocally. However, upon phosphorylation by PKC, MARCKS releases the actin or CaM. MARCKS may therefore act as a CaM sink in resting cells and regulate CaM availability during cell activation. We have demonstrated previously that thrombin-induced myosin light chain (MLC) phosphorylation and increased monolayer permeability in bovine pulmonary artery endothelial cells (BPAEC) require both PKC- and CaM-dependent pathways. We therefore decided to investigate the phosphorylation of MARCKS in BPAEC to ascertain whether this occurs in a temporally relevant manner to participate in the thrombin-induced events. MARCKS is phosphorylated in response to thrombin with a time course similar to that seen with MLC. As expected, MARCKS is also phosphorylated by phorbol 12-myristate 13 acetate (PMA), a PKC activator, but with a slower onset and more prolonged duration. Bradykinin also enhances MARCKS phosphorylation in BPAEC, but histamine does not. MARCKS is distributed evenly between the membrane and cytosol in BPAEC, and neither thrombin nor PMA caused significant translocation of the protein. Specific PKC inhibitors attenuated MARCKS phosphorylation by either thrombin or PMA. Since thrombin-induced MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contraction. W7, a CaM antagonist, enhances the phosphorylation of MARCKS. This was expected since CaM binding to MARCKS has been shown to decrease MARCKS phosphorylation by PKC. On the other hand, tyrosine kinase inhibitors, genistein and tyrphostin, attenuate MARCKS phosphorylation but have no effect on MLC phosphorylation, suggesting that MARCKS may be phosphorylated by kinases other than PKC. Phosphorylation of MARCKS outside the PKC phosphorylation domain would not be expected to induce the release of CaM. These data provide support for the hypothesis that MARCKS may serve as a regulator of CaM availability in BPAEC. © 1996 Wiley-Liss, Inc.     

Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells.

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.     

Inositol phospholipid turnover and protein kinase C translocation are stimulated by poly(I).poly(C) in human amnion cells (UAC)

Polyinosinic-polycytidylic acid, a potent inducer of inducer of interferon (IFN) production and activator of some IFN-induced enzymes, inhibits [3H]uridine incorporation into the RNA of vesicular stomatitis virus even in the absence of IFN synthesis, transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C. Since IFNs also have similar activities these results suggest that IFN induction and IFN function are realised through common biochemical pathways.     

Cooperative interactions of protein kinase C and cAMP-dependent protein kinase systems in human promyelocytic leukemia HL60 cells

Interactions of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) systems were investigated in HL60 cells. It was found that the differentiating effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) were potentiated by dibutyryl cAMP (dbcAMP) or prostaglandin E2 (PGE2). In addition, dbcAMP or PGE2 inhibited TPA-induced binding of PKC to plasma membrane, leading to decreased protein phosphorylation, and promoted subsequent redistribution of enzyme to the nuclear membrane region. The findings are consistent with the hypothesis that PKC and PKA systems regulate cooperatively the phenotypical differentiation of leukemic cells.     

Thrombin inhibits proliferation of the human megakaryoblastic MEG-01 cell line: a possible involvement of a cyclic-AMP dependent mechanism.

Thrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2 synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG-01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG-01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII-related-antigen and cell-size measurement, which are specific markers for megakaryocyte maturation. In addition, incubation of MEG-01 cells with thrombin resulted in dose-dependent increases in cAMP levels, and in inositol-trisphosphate formation and intracellular Ca2+ levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin-induced calcium increase without affecting thrombin-induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG-01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1 and Bt2cAMP. These results suggest the involvement of a cAMP-dependent mechanism in the thrombin-induced reduction in MEG-01 cell growth.     

Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells

Short-term regulation of sodiummetabolism is dependent on the modulation of the activity of sodiumtransporters by first and second messengers. In understanding diseasesassociated with sodium retention, it is necessary to identify thecoupling between these messengers. We have examined whether dopamine,an important first messenger in tubular cells, activates andtranslocates various protein kinase C (PKC) isoforms. We used aproximal tubular-like cell line, LLCPK-1 cells, in which dopamine wasfound to inhibit Na⁺-K⁺-ATPase in aPKC-dependent manner. Translocation of PKC isoforms was studied withboth subcellular fractionation and confocal microscopy. Both techniquesrevealed a dopamine-induced translocation from cytosol to plasmamembrane of PKC- and -, but not of PKC-, -, and -. Theprocess of subcellular fractionation resulted in partial translocationof PKC-. This artifact was eliminated in confocal studies. Confocalimaging permitted detection of translocation within 20 s.Translocation was abolished by a phospholipase C inhibitor and by anantagonist against the dopamine 1 subtype (D₁) but not the2 subtype of receptor (D₂). In conclusion, this studyvisualizes in renal epithelial cells a very rapid activation of thePKC- and - isoforms by the D₁ receptor subtype.

     

IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down-regulation in murine B cells

Co-stimulation of B lymphocytes with IL-4 plus nonmitogenic concentrations of anti-Ig antibodies, or protein kinase C (PKC) activators, drives resting B cells into DNA synthesis. Although cross-linking of the sIg receptors provokes the generation of the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, the molecular mechanism utilized by IL-4R in murine B cells has not, as yet, been defined. In human B cells IL-4 has been shown to induce a transient rise in IP3 followed by a sustained elevation of cAMP. However, in murine B cells, IL-4 does not induce the release of IP3, Ca2+ mobilization, PKC translocation, or indeed modify signaling via the phosphoinositide pathway induced by ligation of sIg receptors. We now present evidence that, in murine B cells, IL-4 synergizes with nonmitogenic concentrations of anti-Ig to provoke translocation of PKC from the cytosol to membranes. In addition, the lymphokine up-regulates PKC levels and activity and prevents phorbol ester-induced PKC down-regulation in B cells. We therefore propose that (unknown) signals generated via IL-4R potentiate and/or prolong sIg-induced PKC activation. These observations may therefore provide a biochemical basis for explaining how IL-4 and anti-Ig synergize to induce B cell activation.     

Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.     

Immunocytochemical evidence for phorbol ester-induced protein kinase C translocation in HL60 cells

The ability of tumor promoting 12-O-tetradecanoylphorbol-13-acetate (TPA) to redistribute protein kinase C in human promyelocytic leukemic HL60 cells was investigated. It was found that TPA caused a rapid translocation (within 10 min) of protein kinase C from the cytosolic (soluble) fraction to the particulate (membrane) fraction, as determined indirectly by assaying for the enzyme activity or by immunoblotting of the enzyme protein in the isolated subcellular fractions. Immunocytochemical localization of the enzyme demonstrated directly that the TPA caused an enzyme translocation t the plasma membrane. These findings suggest that translocation to the plasma membrane of the enzyme may represent initial events related to the TPA effect on terminal differentiation of HL60 cells to monocytes/macrophages.     

Rapid microassay for protein kinase C translocation in Swiss 3T3 cells

The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed to regulate the activity of this kinase [Nishizuka, Y. (1984) Nature (London) 308, 693-698]. A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone H1 was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2 alpha facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells.     

Possible involvement of microfilaments in protein kinase C translocation

We investigated the role of microfilaments in stimulus-induced translocation of protein kinase C (PKC) in polymorphonuclear leukocytes (PMNs) from C57BL/6 mice. Cytochalasin B and dihydrocytochalasin B almost completely inhibited PKC translocation induced by either TPA or Ca2+ ionophore after pretreatment of cells for 30 min. In addition, ML-9, a potent inhibitor of Ca2+/calmodulin-dependent myosin light chain kinase which regulate microfilament contraction, and a calmodulin antagonist W-7, also inhibited PKC translocation. These findings suggest the possibility that microfilaments are involved in the translocation of PKC.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. Evidence for protein kinase C translocation to phagosomes.

This study has investigated the role of protein kinase C (PKC) activation in IgG-mediated phagocytosis by human monocytes. Incubation of monocytes with IgG-opsonized targets increased membrane-associated PKC approximately 2-fold. Kinetic studies showed that the translocation of PKC to membrane occurred before significant ingestion took place. The pharmacologic PKC inhibitor H7 inhibited IgG-dependent ingestion with ID50 of 20 microM, while the structurally related isoquinoline sulfonamide HA1004 had no effect at this concentration. Staurosporine and calphostin C, PKC inhibitors which have different mechanisms of actions than H7, also inhibited ingestion. Depletion of PKC by prolonged incubation with phorbol esters also inhibited phagocytosis, and dose-response curves showed a strong correlation between the extent of PKC depletion and the extent of inhibition of ingestion. Finally, phagosomes were isolated by sucrose density centrifugation of cells disrupted 5 min after the initiation of phagocytosis. Measurement of PKC activity and immunoreactivity in the phagosomes showed that PKC was concentrated in the phagosome membrane approximately 5-fold compared to the uninvolved plasma membrane. Together, these data suggest that PKC activation is an early, essential step in the efficient ingestion of IgG-opsonized targets by monocytes.