Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4183-4194 ◽  
Author(s):  
V Evangelista ◽  
S Manarini ◽  
S Rotondo ◽  
N Martelli ◽  
R Polischuk ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Inhibitory Antibody ◽  
Mixed Cell ◽  
Platelet Surface ◽  
Kinase Activation ◽  
Dynamic Conditions ◽  
Activated Platelets ◽  
Beta 2 ◽  
Adhesion Of Platelets

Abstract Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

scholarly journals Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4183-4194 ◽  
Author(s):  
V Evangelista ◽  
S Manarini ◽  
S Rotondo ◽  
N Martelli ◽  
R Polischuk ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Inhibitory Antibody ◽  
Mixed Cell ◽  
Platelet Surface ◽  
Kinase Activation ◽  
Dynamic Conditions ◽  
Activated Platelets ◽  
Beta 2 ◽  
Adhesion Of Platelets

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 876-885 ◽  
Author(s):  
Virgilio Evangelista ◽  
Stefano Manarini ◽  
Rita Sideri ◽  
Serenella Rotondo ◽  
Nicola Martelli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinase Inhibitors ◽  
Polymorphonuclear Leukocyte ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
Mixed Cell ◽  
Β2 Integrin ◽  
Activated Platelets

Abstract Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 876-885 ◽  
Author(s):  
Virgilio Evangelista ◽  
Stefano Manarini ◽  
Rita Sideri ◽  
Serenella Rotondo ◽  
Nicola Martelli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinase Inhibitors ◽  
Polymorphonuclear Leukocyte ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
Mixed Cell ◽  
Β2 Integrin ◽  
Activated Platelets

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Oncostatin M regulates endothelin-1 production in human endothelial cells

1998 ◽  
Vol 275 (2) ◽  
pp. H662-H667 ◽  
Author(s):  
Outi Saijonmaa ◽  
Tuulikki Nyman ◽  
Päivi Pacek ◽  
Frej Fyhrquist
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Oncostatin M ◽  
Human Umbilical Cord ◽  
Kinase Activation ◽  
Endothelin 1 ◽  
Umbilical Cord Vein ◽  
Pkc Activation ◽  
Human Umbilical Cord Vein

The effect of the macrophage- and T-lymphocyte-derived cytokine oncostatin M (OSM) on endothelin-1 (ET-1) production in cultured human umbilical cord vein endothelial cells (HUVEC) was studied. OSM (2.5–10 ng/ml) stimulated ET-1 production and the expression of preproendothelin-1 mRNA. The stimulatory effect of OSM was reversed by anti-interleukin (IL)-6 IgG (33 μg/ml). IL-6 (10 ng/ml) was shown to stimulate ET-1 production. The tyrosine kinase inhibitors herbimycin (250–500 ng/ml) and genistein (1–4 μg/ml) suppressed basal ET-1 production and reversed the stimulatory effect of OSM, whereas daidzein (1–8 μg/ml), a less active analog of genistein, had no effect on basal ET-1 production and only partly reversed the stimulatory effect of OSM. The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibited ET-1 production. Downregulation of protein kinase C (PKC) with PMA (1 μM) preincubation potentiated OSM-induced ET-1 production. In summary, OSM stimulated ET-1 production in cultured HUVEC. The stimulation was probably mediated by IL-6. Furthermore, the present data suggest that tyrosine kinase activation was involved in ET-1 stimulation and that PKC activation leads to suppression of basal and OSM-stimulated ET-1 production.

scholarly journals Transmission of growth cone traction force through apCAM–cytoskeletal linkages is regulated by Src family tyrosine kinase activity

2001 ◽  
Vol 155 (3) ◽  
pp. 427-438 ◽  
Author(s):  
Daniel M. Suter ◽  
Paul Forscher
Keyword(s):  
Tyrosine Kinase ◽  
Growth Cone ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Traction Force ◽  
Tyrosine Kinase Activity ◽  
Cellular Mechanisms ◽  
Traction Forces ◽  
Kinase Activation ◽  
Neuronal Growth Cone

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor–cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM–cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.

scholarly journals Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

2015 ◽  
Vol 112 (22) ◽  
pp. 6955-6960 ◽  
Author(s):  
Hsin-Yung Yen ◽  
Ying-Chih Liu ◽  
Nai-Yu Chen ◽  
Chia-Feng Tsai ◽  
Yi-Ting Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cancer Progression ◽  
Conformational Changes ◽  
Kinase Inhibitors ◽  
Growth Factor Receptor ◽  
Kinase Activation ◽  
Downstream Signaling ◽  
Egfr Mutant

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.

Coronary arteriolar flow-induced vasodilation signals through tyrosine kinase

1996 ◽  
Vol 270 (6) ◽  
pp. H1878-H1884 ◽  
Author(s):  
J. M. Muller ◽  
M. J. Davis ◽  
W. M. Chilian
Keyword(s):  
Substance P ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Fluorescein Isothiocyanate ◽  
Intraluminal Pressure ◽  
Kinase Activation ◽  
Genistein Treatment ◽  
Dependent Flow ◽  
Coronary Arterioles

Coronary arterioles demonstrate flow-dependent vasodilation that is mediated by endothelial release of nitric oxide. The signaling mechanisms for this response remain unknown. Because tyrosine kinases are an enzyme family linked to many signaling pathways, including some for mechanosensitive transduction, we hypothesized that tyrosine kinase activation is a critical step in flow-induced vasodilation. To test this hypothesis, coronary arterioles were isolated, cannulated with micropipettes, and perfused by two independent reservoir systems. Intraluminal pressure was set at 60 cmH2O, and flow was generated by changing the heights of the reservoirs in equal and opposite directions, thus establishing a pressure difference across the arteriole without altering intraluminal pressure. Vasodilatory responses to intraluminal flow and substance P (1 x 10(-12) to 1 x 10(-7) M) were evaluated before and after intraluminal application of the tyrosine kinase inhibitors genistein (5 microM) and piceatannol (10 microM). Exposure to these inhibitors did not alter spontaneous tone. Substance P caused dose-dependent vasodilation that was not affected by genistein or piceatannol. Increases in intraluminal flow (generated by pressure differences ranging from 4 to 60 cmH2O) elicited graded increases in diameter. Both genistein and piceatannol inhibited the vasodilatory responses to flow. Treatment with daidzein, an inactive analogue of genistein, had no effect on either the flow-induced responses or substance P-induced vasodilation. To further confirm that tyrosine kinase activation is involved in flow-induced vasodilation, vessels were exposed to flow in the absence or presence of genistein and subsequently stained with a fluorescein isothiocyanate-labeled phosphotyrosine antibody. Exposure to flow significantly increased fluorescence of endothelial cells. Genistein treatment reversed the flow-induced increase in tyrosine phosphorylation. These results indicate that endothelium-dependent, flow-induced vasodilation in isolated porcine coronary arterioles is accompanied by an increase in tyrosine kinase activity. We conclude that endothelium-dependent, nitroxidergic, flow-induced vasodilation is mediated, at least in part, by a signaling pathway involving a tyrosine kinase.

The Platelet Glycoprotein IIb/IIIa Complex Is lnvolved in the Adhesion of Activated Platelets to Leukocytes

1993 ◽  
Vol 70 (03) ◽  
pp. 514-521 ◽  
Author(s):  
Peter Spangenberg ◽  
Helge Redlich ◽  
Iris Bergmann ◽  
Wolfgang Lösche ◽  
Matthias Götzrath ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polymorphonuclear Leukocytes ◽  
Surface Membrane ◽  
Rosette Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Normal Expression ◽  
Adhesion Of Platelets

SummaryThe adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counterreceptor (CD 15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein III a which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

scholarly journals Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

2019 ◽  
Author(s):  
Daniel P. Duarte ◽  
Allan J. Lamontanara ◽  
Giuseppina La Sala ◽  
Sukyo Jeong ◽  
Yoo-Kyoung Sohn ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitors ◽  
Md Simulations ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Loss Of Function ◽  
Kinase Activation ◽  
Allosteric Interactions ◽  
B Cell Maturation

ABSTRACTBruton’s tyrosine kinase (Btk) is a key component for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, constitutive Btk signaling drives several B-cell neoplasms, which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the molecular mechanism by which a subset of XLA mutations in the SH2 domain strongly perturbs Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain to which multiple XLA mutations map and which is required for Btk activation. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the Btk SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevented activation of wild-type and TKI-resistant Btk, inhibited Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

scholarly journals Mitogenic Response of Murine B Lymphocytes to Salmonella typhimurium Lipopolysaccharide Requires Protein Kinase C-Dependent Late Tyrosine Phosphorylations

1998 ◽  
Vol 66 (6) ◽  
pp. 2547-2552 ◽  
Author(s):  
Anne Mey ◽  
Jean-Pierre Revillard
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Cross Linking ◽  
Kinase Activation ◽  
Herbimycin A ◽  
Kinase C

ABSTRACT Unlike the cross-linking of membrane immunoglobulins, the activation of B cells by lipopolysaccharide (LPS) does not involve the phosphoinositol turnover and the initial activation of tyrosine kinases. However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture. Tyrosyl-phosphorylated proteins were detected by Western blotting after 24 h of culture with LPS, reaching a maximum concentration after 72 h. Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M antibody and were abrogated by the protein synthesis inhibitor cycloheximide, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine. The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+mobilization and was confirmed by detection of a comparable but restricted pattern of tyrosine-phosphorylated substrates in B cells treated with phorbol myristate acetate alone or in association with ionomycin. Tyrosine kinase activation was dependent on de novo protein synthesis. However, culture supernatants of LPS-activated B cells were devoid of mitogenic activity and induced a phosphorylation pattern more restricted than that achieved by LPS. Altogether these data indicate that proliferation signals induced by LPS or by the cross-linking of membrane immunoglobulins are controlled by late tyrosine phosphorylations occurring throughout the first 3 days of culture, controlled in part by protein kinase C activation, and dependent on the synthesis of an intermediate protein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.

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