FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2069720072> ?p ?o ?g. }

• W2069720072 endingPage "30315" @default.
• W2069720072 startingPage "30306" @default.
• W2069720072 abstract "Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical ζ protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous ζ protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of ζ protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks ζ but not α protein kinase C plasma membrane translocation. Synthetic peptides from ζ protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of ζ but not α protein kinase C. These data suggest the involvement of ζ protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation. Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical ζ protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous ζ protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of ζ protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks ζ but not α protein kinase C plasma membrane translocation. Synthetic peptides from ζ protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of ζ but not α protein kinase C. These data suggest the involvement of ζ protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation. formyl-Met-Leu-Phe interleukin-8 diacylglycerol protein kinase C phorbol myristate acetate phosphate-buffered saline fetal calf serum. Leukocyte extravasation is crucial for an appropriate and effective immune response. Interaction with vascular endothelium is a carefully regulated multistep process leading to selective migration of various leukocyte subtypes. Adhesion molecules and activating factors control different steps of this process. Selectins and α4-integrins both mediate the initial interaction and the subsequent rolling along the endothelium, whereas only integrins mediate firm adhesion (1Butcher E.C. Picker L.J. Science. 1996; 272: 60-66Crossref PubMed Scopus (2519) Google Scholar). The transition from rolling to firm adhesion requires intracellular biochemical changes, since integrins do not recognize the ligand unless activated. Several factors have been reported to trigger activation of integrin-dependent leukocyte adhesion and motility, including phorbol esters, cytokines, and chemoattractants (2Patarroyo M. Yogeeswaran G. Biberfeld P. Klein E. Klein G. Int. J. Cancer. 1982; 30: 707-717Crossref PubMed Scopus (47) Google Scholar, 3Von Andrian U.H. Hansell P. Chambers J.D. Berger E.M. Torres Filho I. Butcher E.C. Arfors K.E. Am. J. Physiol. 1992; 263: 1034-1044Crossref PubMed Google Scholar, 4Ley K. Baker J.B. Cybulsky M.I. Gimbrone Jr., M.A. Luscinskas F.W. J. Immunol. 1993; 151: 6347-6357PubMed Google Scholar). Pertussis toxin-induced inhibition of lymphocyte homing highlighted the role of Gαi-protein linked receptors and their ligands as physiological activators of integrin-dependent lymphocyte adhesion in vivo(5Bargatze R.F. Butcher E.C. J. Exp. Med. 1993; 178: 367-372Crossref PubMed Scopus (252) Google Scholar). Classical chemoattractants, such as formyl-Met-Leu-Phe (fMLP),1 and chemokines, such as interleukin 8 (IL-8), trigger α4β1-integrin-dependent lymphocyte adhesion to vascular cell adhesion molecule-1 (VCAM-1) and αMβ2-integrin-dependent polymorphonuclear neutrophil adhesion to fibrinogen through a Pertussis toxin-sensitive signaling pathway previously shown to involve Rho small GTP-binding proteins (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). A well known signaling cascade triggered by chemoattractants involves activation of phosopholipase C, leading to diacylglycerol (DAG) and inositol triphosphate accumulation, intracellular calcium increase, and activation of the serine-threonine protein kinase C (PKC) (7Gerard G. Gerard G. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar). PKC is a family of closely related proteins with serine-threonine kinase activity subdivided in subfamilies according to their sensitivity to Ca2+ and DAG. These include the classical, Ca2+-DAG sensitive, isozymes (α, βI, βII, and γ PKC); the novel, Ca2+--independent but DAG-sensitive, isozymes (δ, ε, η, and θ PKC); and the atypical, Ca2+- and DAG-independent, isozymes (ζ and λ/ι PKC). Involvement of PKC in integrin activation has been originally suggested by the capability of the DAG-synthetic analog phorbol myristate acetate (PMA), a direct activator of DAG-sensitive PKCs, to activate integrin-dependent leukocyte aggregation and adhesion (2Patarroyo M. Yogeeswaran G. Biberfeld P. Klein E. Klein G. Int. J. Cancer. 1982; 30: 707-717Crossref PubMed Scopus (47) Google Scholar). However, PMA is not a physiological agonist, and in studies reported earlier (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar), we showed that chemoattractant activation of rapid integrin-dependent leukocyte adhesion was not affected by calphostin C, an inhibitor of DAG-sensitive PKC isozymes that interferes with DAG binding to the C1 regulatory domain (8Kobayashi E. Nakano H. Morimoto M. Tamaoki T. Biochem. Biophys. Res. Commun. 1989; 159: 548-553Crossref PubMed Scopus (1072) Google Scholar). To clarify the involvement of PKC in integrin activation and chemotaxis triggering by chemoattractants in human polymorphonuclear neutrophils, we used PKC inhibitors able to discern among different subfamilies of isozymes. Particularly, we exploited the highly selective inhibitory activity of synthetic peptides with sequence derived from the N-terminal pseudosubstrate region of PKC (9House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (791) Google Scholar). We show evidence suggesting that Ca2+-DAG-sensitive PKC isoforms are not involved in the chemoattractant-triggered signaling pathway leading to integrin-dependent neutrophil adhesion and chemotaxis. In contrast, inhibitors of the atypical ζ PKC block adhesion and chemotaxis induced by chemoattractants as well as adhesion triggered by phorbol esters. Inhibitors of the atypical ζ PKC also block chemoattractant-induced G-actin assembly. Moreover, chemoattractant and PMA-induced plasma membrane translocation of ζ PKC is selectively blocked by C3 transferase, a specific inhibitor of Rho small GTP-binding protein. Thus, our data suggest a role of ζ PKC as downstream effector of signaling pathways leading to integrin activation and movement in human neutrophils. Furthermore, a potential functional link with Rho small GTPases is proposed. Human blood polymorphonuclear neutrophils were isolated by dextran sedimentation and centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech) as described previously (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). Contaminating erythrocytes were lysed by hypotonic saline, and then neutrophils were washed with PBS and finally resuspended in RPMI 1640 containing 10% FCS. All of the above procedures were done using reagents prepared in endotoxin-free water for clinical use. All peptides (synthesized at the Stanford University Protein and Nucleic Acid Facility) were solubilized immediately before use at a 1 mm concentration in PBS, pH 7.2. In some cases (myristoylated ζ PKC short and long) peptides were heated to 37–40 °C to achieve complete solubility. Reported below are peptide sequences from the pseudosubstrate region of human PKC isozymes: α PKC long, RFARKGALRQKNVHEVK (positions 19–35); α PKC short, RFARKGALRQKNV (positions 19–31); α PKC scramble short, LVFQGKERVARRNHKA; α PKC scramble long, LFQGKRVARRNA; ζ PKC long, SIYRRGARRWRKLYRAN (positions 113–129); ζ PKC short, SIYRRGARRWRKL (positions 113–125); ζ PKC scramble long, RLRYRNKRIWRSAYAGR; ζ PKC scramble short, RLYRKRIWRSAGR. PBS, fMLP, PMA, leupeptin, pepstatin, aprotinin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and human fibrinogen were purchased from Sigma; diisopropyl fluorophosphate was from Aldrich; RPMI 1640 and FCS were from Irvine; antibodies against PKC isozymes were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Transduction Laboratories; calphostin C, Gö6976, Gö6850 (bisindolylmaleimide I), and chelerythrine chloride were from Alexis Biochemicals (San Diego, CA); recombinant C3 transferase was from Upstate Biotechnology Inc. (Lake Placid, NY); dihydrorhodamine and nitrobenzoxadiazole/phallacidin were from Molecular Probes (Eugene, OR); unlabeled phallacidin was from Sigma; horseradish peroxidase was from Sigma; trisacryl protein A was from Pierce; and myelin basic protein was from Sigma. Eighteen-well glass slides were coated for 120 min at 37 °C with human fibrinogen (Sigma) (20 μg/well in endotoxin-free PBS). Neutrophils (5 × 104/well; 2.5 × 106/ml in RPMI 1640, containing 10% heat-inactivated FCS and 20 mm HEPES, pH 7.3) were added, incubated for 10 min at 37 °C, and then stimulated by the addition of the agonists before washing, fixation on ice in 1.5% glutaraldehyde for 60 min, and computer-assisted enumeration of cells bound in 0.2 mm2, as described (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). Neutrophils migration was assessed using 1-μm pore size transwells (Bio-Coat, Becton Dickinson). Neutrophil were in RPMI 1640, containing 10% heat-inactivated FCS and 20 mm HEPES, pH 7.3, at 2 × 106/ml. 100 μl of cell suspension were added to the top well, and 600 μl of medium, containing agonists and inhibitors, were added to the bottom well. After fixation with 1.5% glutaraldehyde, migrated cells were counted by fluorescence-activated cell sorting using polystyrene beads (Polyscience) as an internal standard (10Campbell J.J. Qin S. Bacon K.B. Mackay C.R. Butcher E.C. J. Cell Biol. 1996; 134: 255-266Crossref PubMed Scopus (208) Google Scholar) Activation of neutrophil NADPH-oxidase was evaluated by measuring reduction of dihydrorhodamine induced by superoxide anion-derived hydrogen peroxide, as previously reported (11Vowells S.J. Sekhsaria S. Malech H.L. Shalit M. Fleisher T.A. J. Immunol. Methods. 1995; 178: 89-97Crossref PubMed Scopus (396) Google Scholar, 12Henderson L.M. Chappell J.B. Eur. J. Biochem. 1993; 217: 973-980Crossref PubMed Scopus (316) Google Scholar). Neutrophils, resuspended at 2 × 104/ml in PBS, pH 7.3, containing 1 mmCaCl2, 1 mm MgCl2, 20 μg/ml horseradish peroxidase, 0.5 μm dihydrorhodamine (reaction buffer), were stimulated under stirring at 37 °C. The time course of hydrogen peroxide-induced dihydrorhodamine reduction was evaluated with a spectrofluorimeter with 505 of excitation wavelength and 534 of emission wavelength. Calibration was done using as a standard a defined amount of hydrogen peroxide. F-actin was quantified by methanol extraction of nitrobenzoxadiazole/phallacidin-stained cells, as described previously (66Howard T.H. Oresajo C.O. Cell Motil. 1985; 6: 545-557Crossref Scopus (90) Google Scholar). Neutrophils (5 × 106) were pretreated on ice for 30 min with 30 μg/ml IB4 mouse monoclonal antibody anti-human β2-integrins (Fab2fragment) to prevent agonist-triggered integrin-mediated neutrophil aggregation and signaling. The cells were then washed; resuspended in PBS, pH 7.3, containing 1 mm CaCl2, 1 mm MgCl2; stimulated under stirring at 37 °C; fixed with 2% paraformaldehyde; and stained for 30 min at room temperature in PBS containing 0.1% Triton X-100 and 0.4 μm nitrobenzoxadiazole/phallacidin. After sedimentation at 16,000 × g, the pellet was overlaid with 0.5 ml of absolute methanol and extracted for 6 h in the dark at 4 °C. After cell sedimentation, the supernatant was collected, and the fluorescence intensity was determined with a spectrofluorimeter (excitation at 465 nm; emission at 535 nm). To determine the level of nonspecific binding, a 10-fold molar excess of unlabeled phallacidin was included. The value of nonspecific binding was subtracted to yield specific F-actin staining and quantification. Neutrophils were pretreated with diisopropyl fluorophosphate (5.8 mm for 20 min on ice) and subsequently pretreated on ice for 30 min with 30 μg/ml IB4 mouse monoclonal antibody anti-human β2-integrins (Fab2 fragment) to prevent agonist-triggered integrin-mediated neutrophil aggregation and signaling. Neutrophils were then washed, resuspended at 4 × 107/ml in PBS containing 1 mm Ca2+/Mg2+, and stimulated with agonists under stirring at 37 °C. Stimulation was stopped with lysis buffer containing 50 mm Tris-HCl, pH 7.5; 1% Triton X-100; 0.01% SDS; 150 mm NaCl; 50 mm NaF; 10 mm sodium pyrophosphate; 2 mm EDTA; 1 mm EGTA; 1 μmdithiothreitol; 1 μm phenylarsine oxide; 1 mmphenylmethylsulfonyl fluoride; 2.5 mm benzamidine; and 20 μg/ml of leupeptin, pepstatin, aprotinin, and soybean trypsin inhibitor. After 30 min on ice, lysates were centrifuged at 16,000 × g for 1 min to remove cell debris. Rabbit polyclonal anti-ζ PKC (1 μg) or control rabbit serum was added to an equal amount of cell lysates, followed by immunoprecipitation with trisacryl protein A. Equal amounts of ζ PKC were immunoprecipitated as confirmed by Western blot analysis (data not shown). After four washings, immunoprecipitates were subjected to the kinase reaction for 30 min at 30 °C in 50 μl of kinase buffer containing 0.5 mm EGTA, 10 mm MgCl2, 20 mm HEPES, pH 7.4, 50 μm ATP, 5 μCi of [γ-32P]ATP, and 2 μg of myelin basic protein as a substrate. The reaction was stopped by the addition of 5% trichloroacetic acid, and the reaction mixture was filtered through phosphocellulose paper. After four rinses with 1% phosphoric acid radioactivity on the filter was determined with a scintillation counter. Neutrophils were pretreated with diisopropyl fluorophosphate (5.8 mm for 20 min on ice) and subsequently electroporated in the presence of 25 μg/ml of recombinant C3 transferase, as previously reported (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). The effectiveness of the treatment was tested every time by evaluating C3 transferase-induced inhibition of triggered adhesion, as previously reported (6Laudanna C. Campbel J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar) (data not shown). After C3 treatment, neutrophils were pretreated on ice for 30 min with 30 μg/ml of IB4 mouse monoclonal antibody anti-human β2-integrins (Fab2fragment) to prevent agonist-triggered integrin-mediated neutrophil aggregation and signaling. Neutrophils were then washed, resuspended at 3 × 107/ml in PBS containing 1 mmCa2+/Mg2+, and stimulated under stirring with agonists at 37 °C. Stimulation was stopped by diluting the cells in a 10 times larger volume of ice-cold PBS. Cells, resuspended in 1 ml of ice-cold PBS containing 8% sucrose, 1 mmphenylmethylsulfonyl fluoride, and 20 μg/ml leupeptin, pepstatin, aprotinin, soybean trypsin inhibitor were sonicated, and the homogenates were centrifuged at 800 × g for 10 min to remove nuclei and unbroken cells. The postnuclear supernatant was loaded on a discontinuous sucrose gradient (50% sucrose, 30% sucrose) and centrifuged for 90 min at 100,000 × g. The light membrane fraction (plasma membrane) was collected in the 30% layer (13Quinn M.T. Parkos C.A. Walker L. Orkin S.H. Dinauer M.C. Jesaitis A.J. Nature. 1989; 342: 198-200Crossref PubMed Scopus (181) Google Scholar). Following SDS-polyacrylamide gel electrophoresis on 9% acrylamide, proteins (10 μg/lane) were electroblotted on 0.2-μm pore size nitrocellulose filters, probed with rabbit anti-α or -ζ PKC polyclonal antibodies (Santa Cruz Biotechnology), followed by goat polyclonal anti-rabbit horseradish peroxidase conjugated (Sigma) and developed using ECL (Amersham Pharmacia Biotech). The specificity of the anti-ζ PKC antibodies was confirmed by the capability to recognize a unique band of approximately 72 kDa on blotted nitrocellulose filters of either rat brain lysates or baculovirus-purified human recombinant ζ PKC (Panvera) (data not shown). The role of PKC in neutrophil integrin activation and chemotaxis was initially investigated using various PKC inhibitors. As previously reported, calphostin C did not block fMLP or IL-8-triggered neutrophil adhesion to fibrinogen. Gö6976, a structurally unrelated PKC inhibitor with selectivity for classical (α, β, and γ) isozymes of PKC (14Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marmé D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), did not interfere with chemoattractant-induced adhesion. Gö6850 (bisindolylmaleimide I), a third structurally unrelated inhibitor of classical and novel (δ, ε, η, and θ) PKC (14Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marmé D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), had no detectable effect on chemoattractant-induced neutrophil adhesion. (Fig. 1 A). In contrast, adhesion stimulated by the phorbol ester PMA, a direct activator of DAG-dependent PKC isozymes, was effectively blocked by all these inhibitors (Fig. 1 A). Staurosporine, a potent but relatively nonselective kinase inhibitor, also inhibited PMA but not chemoattractant-induced neutrophil adhesion (data not shown). Moreover, analysis of neutrophil chemotaxis showed no influence of Gö6976 and Gö6850 on neutrophil migration toward the chemoattractants fMLP and IL-8 (Fig. 1 B); staurosporine was also ineffective (data not shown). Because of its reported light dependence (15Burns R.F. Miller F.D. Merriman R.L. Howbert J.J. Heath W.F. Kobayashi E. Takahashi I. Tamaoki T. Nakano H. Biochem. Biophys. Res. Commun. 1991; 176: 288-293Crossref PubMed Scopus (359) Google Scholar), the effect of calphostin C on chemotaxis was not evaluated. These data clearly suggest that chemoattractant activation of neutrophil integrin-dependent adhesion and chemotaxis does not involve DAG-sensitive PKC isozymes. To verify the previous data, we used Chelerythrine chloride, a specific (but not isozyme-selective) PKC inhibitor that represents a unique class of PKC inhibitors that competitively interfere with the phosphate acceptor site and noncompetitively inhibits the ATP binding site (16Herbert M.J. Augereu J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Crossref PubMed Scopus (1196) Google Scholar). Surprisingly, pretreatment of neutrophils with chelerythrine chloride efficiently blocked fMLP, IL-8, and PMA-induced adhesion in a dose-dependent manner (Fig. 2 A). Moreover, chelerythrine chloride also inhibited chemotaxis to both agonists in a dose-dependent manner (Fig. 2 B). We conclude that a chelerythrine chloride-sensitive but calphostin C-, Gö6976-, Gö6850-, and staurosporine-insensitive PKC isoform is involved in chemoattractant triggering of integrin-dependent neutrophil adhesion and chemotaxis. Importantly, none of the inhibitors affected expression of β2-integrins, cell viability as assessed by trypan blue exclusion, or intracellular Ca2+ elevation stimulated by fMLP or IL-8 (data not shown). To further address these findings, we took advantage of the ability of synthetic pseudosubstrate peptides, with sequences based on the endogenous PKC pseudosubstrate region (9House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (791) Google Scholar,17Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1218) Google Scholar), to inhibit PKC kinase activity. Peptides were synthesized with an N-terminal myristic acid to facilitate their diffusion through the plasma membrane, as previously reported. (18Eichholtz T. de Bont D.B.A. de Widt J. Liskamp R.M.J. Ploegh H.L.. J. Biol. Chem. 1993; 268: 1982-1986Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 3 A, myristoylated peptides from α PKC, “short,” encompassing amino acids 19–31 (reported to be a more potent inhibitor (9House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (791) Google Scholar)) and “long,” amino acids 19–35 (encompassing the entire pseudosubstrate region, which is identical in both α and β classical PKC isozymes) had no effect on adhesion stimulated by fMLP and IL-8. In contrast, PMA-induced adhesion was blocked in a dose-dependent manner, whereas nonmyristoylated peptides with the same α PKC sequence and myristoylated peptides with a “scrambled” sequence were ineffective, even at the highest concentrations. Both short and long myristoylated peptides from α PKC were also unable to block neutrophil chemotaxis to fMLP and IL-8 (Fig. 3 B). These data confirm the previous observations and show that myristoylated peptides from PKC pseudosubstrate region are suitable tools to study PKC function in human neutrophils. The previous data show that a DAG-independent PKC isozyme, sensitive to chelerythrine chloride but not to other PKC inhibitors and peptides, is involved in chemoattractant-induced neutrophil adhesion and chemotaxis. Western blot analysis revealed that human neutrophils express PKC isozymes of the classical (α, βI, and βII), novel (δ), and atypical (ζ) subfamilies of PKC, also consistent with previous a report (19Kent J.D. Sergeant S. Burns D.J. McPhail L.C. J. Immunol. 1996; 157: 4641-4647PubMed Google Scholar), whereas in control rat brain lysates ε, η, and λ/ι PKCs were also expressed (data not shown). Thus, the ζ isozyme was the only atypical PKC expressed in human neutrophils. Since the atypical ζ PKC isozyme has been previously shown to be chelerythrine chloride-sensitive (20Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) but insensitive to staurosporine (21Kochs G. Hummel R. Meyer D. Hug H. Marmé D. Sarre T.F. Eur. J. Biochem. 1993; 216: 597-606Crossref PubMed Scopus (86) Google Scholar, 22Kazanietz M.G. Bustelo X.R. Barbacid M. Kolch W. Mishak H. Wong G. Pettit G.R. Bruns J.D. Blumberg P.M. J. Biol. Chem. 1994; 269: 11590-11594Abstract Full Text PDF PubMed Google Scholar, 23Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (471) Google Scholar), we hypothesized that ζ PKC might be a target of chelerythrine chloride inhibition in our model and thus an effector of chemoattractant-triggered signaling pathways leading to integrin-dependent adhesion and chemotaxis. We tested the effect of peptides derived from the pseudosubstrate region of ζ PKC. Two myristoylated peptides from ζ PKC (short, encompassing amino acids 113–125, and long, amino acids 113–129, encompassing the entire pseudosubstrate region) inhibited in a dose-dependent manner fMLP- and IL-8-stimulated adhesion (Fig. 4 A). The long peptide was the most efficient inhibitor, with more than 90% inhibition at 50 μm. Nonmyristoylated peptides with the same ζ PKC sequence and myristoylated peptides with a scrambled sequence were ineffective, even at the highest concentrations (Fig. 4 A). We then asked whether ζ PKC pseudosubstrate peptides might affect chemoattractant-induced chemotaxis. Myristoylated ζ PKC peptides inhibited in a dose-dependent manner fMLP- and IL-8-induced chemotaxis (Fig. 4 B). As shown for the adhesion, the long peptide was the most efficient inhibitor, whereas peptides that were nonmyristoylated or myristoylated with a scrambled sequence were ineffective (Fig. 4 B). Again, cell viability, expression of β2-integrins, and intracellular Ca2+elevation induced by fMLP and IL-8 were not affected by pretreatment with any of the peptides (data not shown). To verify the specificity of the ζ PKC peptides, we tested their effect on PMA-induced adhesion. Surprisingly, myristoylated ζ PKC pseudosubstrate peptides also blocked in dose-dependent manner PMA-triggered adhesion; again the long peptide was the most efficient inhibitor, with more than 85% inhibition at 50 μm. Peptides that were nonmyristoylated or myristoylated with a scrambled sequence were ineffective (Fig. 5 A). This finding was rather unexpected, since the pseudosubstrate regions of α and ζ PKCs are quite different (23% of homology), and ζ PKC is not activated by PMA. We then evaluated the activation of the superoxide-generating NADPH-oxidase system of neutrophils, another function triggered by PMA. Myristoylated ζ PKC pseudosubstrate peptides did not interfere with PMA-triggered NADPH-oxidase activation, even at low doses of PMA and high doses of peptides (Fig. 5 B), whereas myristoylated α PKC pseudosubstrate peptides effectively blocked NADPH-oxidase activation. These data support the selectivity of ζ PKC peptides, suggesting that atypical ζ PKC may be an effector of PMA-triggered signaling pathways leading to integrin activation. Chemoattractants stimulate in neutrophils rapid cytosolic G-actin polymerization leading to accumulation of filamentous actin (F-actin). Although F-actin accumulation does not seem not to be necessary for leukocyte integrin activation, as suggested by the incapacity of cytochalasin B to block lymphocyte function antigen type 1-mediated lymphocyte aggregation (27Tominaga T. Sugie K. Hirata M. Morii N. Fukata J. Uchida A. Imura H.. Narumiya S. J. Cell Biol. 1993; 120: 1529-1537Crossref PubMed Scopus (206) Google Scholar), actin polymerization is likely to be required for leukocyte shape change, such as polarization and directional movement during chemotaxis. Thus, we asked whether ζ PKC could be involved in signaling events leading to G-actin polymerization triggered by chemoattractants. As shown in Fig. 6, fMLP and IL-8 induced rapid increase of F-actin content. Neutrophil pretreatment with chelerythrine chloride or with myristoylated ζ PKC pseudosubstrate peptides almost completely abolished the increase of F-actin. In contrast, peptide myristoylated with a scrambled sequence did not block F-actin accumulation. Thus, ζ PKC seems to be involved in signaling cascade leading to G-actin polymerization. To characterize further ζ PKC involvement in proadhesive signaling pathways, we measured its kinase activity. In nonstimulated neutrophils, ζ PKC showed constitutive kinase activity (Fig. 7), as also previously reported (25Liyanage M. Frith D. Livneh E. Stabel S. Biochem. J. 1992; 283: 781-787Crossref PubMed Scopus (103) Google Scholar). fMLP and IL-8 induced a 4.3- and 2.4-fold increase, respectively, of ζ PKC activity. Consistent with the rapid kinetics of chemoattractant-induced cell adhesion, fMLP- and IL-8-induced increase of ζ PKC activity was very rapid, occurring within 10 s, the earliest time point measurable (Fig. 7). However, increase of ζ PKC activity was transient, with a decrease of about 63% (fMLP) and 51% (IL-8) within 30 s. In contrast, PMA did not induce any significant increase of ζ PKC activity even after 5 min of stimulation (Fig. 7). fMLP and IL-8 but not PMA also triggered an increase of ζ PKC autophosphorylating activity, as measured in a kinase assay carried out in the absence of myelin basic protein (data not shown). Translocation of PKC isozymes to different cellular compartments is a hallmark of selective PKC activation (24Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar), and it is thought to be as important as or more important than altered kinase activity in regulating PKC functions within the cell (24Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar); translocation is likely to be particularly important in regulating ζ PKC function because, unlike classical PKC isoforms, ζ PKC displays a high level of constitutive kinase activity in in vitro assays (Refs. 25Liyana" @default.
• W2069720072 created "2016-06-24" @default.
• W2069720072 creator A5017828443 @default.
• W2069720072 creator A5028728182 @default.
• W2069720072 creator A5035047473 @default.
• W2069720072 creator A5039639608 @default.
• W2069720072 creator A5073711928 @default.
• W2069720072 date "1998-11-01" @default.
• W2069720072 modified "2023-09-29" @default.
• W2069720072 title "Evidence of ζ Protein Kinase C Involvement in Polymorphonuclear Neutrophil Integrin-dependent Adhesion and Chemotaxis" @default.
• W2069720072 cites W129191972 @default.
• W2069720072 cites W1481203098 @default.
• W2069720072 cites W1487365829 @default.
• W2069720072 cites W148785554 @default.
• W2069720072 cites W1503737214 @default.
• W2069720072 cites W1517639955 @default.
• W2069720072 cites W1522933979 @default.
• W2069720072 cites W1529396499 @default.
• W2069720072 cites W1543728556 @default.
• W2069720072 cites W1544321151 @default.
• W2069720072 cites W1553713007 @default.
• W2069720072 cites W1573258139 @default.
• W2069720072 cites W1607258414 @default.
• W2069720072 cites W1618394762 @default.
• W2069720072 cites W1641731911 @default.
• W2069720072 cites W1663295833 @default.
• W2069720072 cites W1818313483 @default.
• W2069720072 cites W1971132834 @default.
• W2069720072 cites W1981023296 @default.
• W2069720072 cites W1995191054 @default.
• W2069720072 cites W1996121260 @default.
• W2069720072 cites W1996668125 @default.
• W2069720072 cites W1998067501 @default.
• W2069720072 cites W2005304515 @default.
• W2069720072 cites W2008323459 @default.
• W2069720072 cites W2012968512 @default.
• W2069720072 cites W2019601714 @default.
• W2069720072 cites W2020993672 @default.
• W2069720072 cites W2023165940 @default.
• W2069720072 cites W2034839112 @default.
• W2069720072 cites W2039117682 @default.
• W2069720072 cites W2042626190 @default.
• W2069720072 cites W2043290332 @default.
• W2069720072 cites W2045891901 @default.
• W2069720072 cites W2050868565 @default.
• W2069720072 cites W2054613918 @default.
• W2069720072 cites W2055104787 @default.
• W2069720072 cites W2056104802 @default.
• W2069720072 cites W2062077009 @default.
• W2069720072 cites W2067393120 @default.
• W2069720072 cites W2075473225 @default.
• W2069720072 cites W2076075128 @default.
• W2069720072 cites W2082607775 @default.
• W2069720072 cites W2083020576 @default.
• W2069720072 cites W2085547973 @default.
• W2069720072 cites W2089330962 @default.
• W2069720072 cites W2091673674 @default.
• W2069720072 cites W2117227310 @default.
• W2069720072 cites W2125070282 @default.
• W2069720072 cites W2135308914 @default.
• W2069720072 cites W2136059362 @default.
• W2069720072 cites W2144420832 @default.
• W2069720072 cites W2148623085 @default.
• W2069720072 cites W2155047359 @default.
• W2069720072 cites W2164809662 @default.
• W2069720072 cites W2167039432 @default.
• W2069720072 cites W2169132737 @default.
• W2069720072 cites W2170057807 @default.
• W2069720072 cites W2402807598 @default.
• W2069720072 cites W2413462302 @default.
• W2069720072 cites W2418425129 @default.
• W2069720072 cites W4211264883 @default.
• W2069720072 doi "https://doi.org/10.1074/jbc.273.46.30306" @default.
• W2069720072 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9804792" @default.
• W2069720072 hasPublicationYear "1998" @default.
• W2069720072 type Work @default.
• W2069720072 sameAs 2069720072 @default.
• W2069720072 citedByCount "228" @default.
• W2069720072 countsByYear W20697200722012 @default.
• W2069720072 countsByYear W20697200722013 @default.
• W2069720072 countsByYear W20697200722014 @default.
• W2069720072 countsByYear W20697200722015 @default.
• W2069720072 countsByYear W20697200722016 @default.
• W2069720072 countsByYear W20697200722017 @default.
• W2069720072 countsByYear W20697200722018 @default.
• W2069720072 countsByYear W20697200722019 @default.
• W2069720072 countsByYear W20697200722020 @default.
• W2069720072 countsByYear W20697200722021 @default.
• W2069720072 countsByYear W20697200722022 @default.
• W2069720072 crossrefType "journal-article" @default.
• W2069720072 hasAuthorship W2069720072A5017828443 @default.
• W2069720072 hasAuthorship W2069720072A5028728182 @default.
• W2069720072 hasAuthorship W2069720072A5035047473 @default.
• W2069720072 hasAuthorship W2069720072A5039639608 @default.
• W2069720072 hasAuthorship W2069720072A5073711928 @default.
• W2069720072 hasBestOaLocation W20697200721 @default.
• W2069720072 hasConcept C1491633281 @default.
• W2069720072 hasConcept C170493617 @default.

• next