Physical and Functional Association of FcαR With Protein Tyrosine Kinase Lyn

In this report, we show that the Src family nonreceptor protein tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor (FcαR) in the monocytic cell line THP-1. Receptor aggregation and subsequent immunoprecipitation of receptor complexes with huIgA adsorbed to nitrocellulose particles shows that Lyn associates with FcαR by a mechanism sensitive to short treatment with the Src family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc receptor (FcγR) in THP-1 cells was unaffected by short treatment with the PTK inhibitor. Cross-linking of FcαR induced tyrosine phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK activity. Unexpectedly, in vitro kinase assays showed that FcαR aggregation-induced tyrosine phosphorylation of Syk did not result in upregulation of Syk activity. Despite the lack of enhanced Syk kinase activity, downstream signaling after FcαR cross-linking was functional and induced the release of significant amounts of interleukin-1 receptor antagonist and interleukin-8. The induction of cytokine release was completely blocked by PP1, thus confirming the biological significance of the association of Lyn with aggregated FcαR. Our data show that early signal transduction after FcαR cross-linking as well as FcαR-mediated activation of cellular effector functions depends on Src family kinase activity. The Src-family PTK involved in FcαR-mediated tyrosine phosphorylation appears to be Lyn, which coprecipitated with aggregated FcαR complexes.

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Physical and Functional Association of FcαR With Protein Tyrosine Kinase Lyn

Blood ◽

10.1182/blood.v91.2.383 ◽

1998 ◽

Vol 91 (2) ◽

pp. 383-391 ◽

Cited By ~ 28

Author(s):

Heinz Gulle ◽

Aysen Samstag ◽

Martha M. Eibl ◽

Hermann M. Wolf

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Biological Significance ◽

Fc Receptor ◽

Cross Linking ◽

Short Treatment ◽

Protein Tyrosine ◽

Receptor Complexes

Abstract In this report, we show that the Src family nonreceptor protein tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor (FcαR) in the monocytic cell line THP-1. Receptor aggregation and subsequent immunoprecipitation of receptor complexes with huIgA adsorbed to nitrocellulose particles shows that Lyn associates with FcαR by a mechanism sensitive to short treatment with the Src family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc receptor (FcγR) in THP-1 cells was unaffected by short treatment with the PTK inhibitor. Cross-linking of FcαR induced tyrosine phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK activity. Unexpectedly, in vitro kinase assays showed that FcαR aggregation-induced tyrosine phosphorylation of Syk did not result in upregulation of Syk activity. Despite the lack of enhanced Syk kinase activity, downstream signaling after FcαR cross-linking was functional and induced the release of significant amounts of interleukin-1 receptor antagonist and interleukin-8. The induction of cytokine release was completely blocked by PP1, thus confirming the biological significance of the association of Lyn with aggregated FcαR. Our data show that early signal transduction after FcαR cross-linking as well as FcαR-mediated activation of cellular effector functions depends on Src family kinase activity. The Src-family PTK involved in FcαR-mediated tyrosine phosphorylation appears to be Lyn, which coprecipitated with aggregated FcαR complexes.

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Activation of c-Src in cells bearing v-Crk and its suppression by Csk

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4706-4713.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4706-4713

Author(s):

H Sabe ◽

M Okada ◽

H Nakagawa ◽

H Hanafusa

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Src Kinase ◽

Cellular Protein ◽

Morphological Transformation ◽

Protein Tyrosine ◽

In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

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The GPI-anchored adhesion molecule F3 induces tyrosine phosphorylation: involvement of the FNIII repeats

Journal of Cell Science ◽

10.1242/jcs.109.3.699 ◽

1996 ◽

Vol 109 (3) ◽

pp. 699-704 ◽

Cited By ~ 1

Author(s):

M. Cervello ◽

V. Matranga ◽

P. Durbec ◽

G. Rougon ◽

S. Gomez

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Cho Cells ◽

Intracellular Signaling ◽

Protein Tyrosine Kinase ◽

Cross Linking ◽

Protein Tyrosine ◽

Intracellular Signaling Pathway ◽

Type Iii ◽

Cell Cell

The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cell-cell recognition. In particular, it is not known whether F3 transduces signals across the membrane. Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-cross-linking. Furthermore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.

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Signal transduction of the human granulocyte-macrophage colony- stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins

Blood ◽

10.1182/blood.v76.4.706.bloodjournal764706 ◽

1990 ◽

Vol 76 (4) ◽

pp. 706-715 ◽

Cited By ~ 1

Author(s):

Y Kanakura ◽

B Druker ◽

SA Cannistra ◽

Y Furukawa ◽

Y Torimoto ◽

...

Keyword(s):

Signal Transduction ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine- kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM- CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM- CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.

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Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils.

The Journal of Cell Biology ◽

10.1083/jcb.126.4.1111 ◽

1994 ◽

Vol 126 (4) ◽

pp. 1111-1121 ◽

Cited By ~ 111

Author(s):

G Berton ◽

L Fumagalli ◽

C Laudanna ◽

C Sorio

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Class I ◽

Human Neutrophils ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Mhc Antigens ◽

Beta 2

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.

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Inhibition by 5′-methylthioadenosine of cell growth and tyrosine kinase activity stimulated by fibroblast growth factor receptor in human gliomas

Journal of Neurosurgery ◽

10.3171/jns.1995.83.4.0690 ◽

1995 ◽

Vol 83 (4) ◽

pp. 690-697 ◽

Cited By ~ 10

Author(s):

Katsuya Miyaji ◽

Eiichi Tani ◽

Atsuhisa Nakano ◽

Hideyasu Ikemoto ◽

Keizo Kaba

Keyword(s):

Growth Factor ◽

Cell Growth ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Glioma Cells ◽

Protein Tyrosine Phosphorylation ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine

✓ Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5′-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor— or platelet-derived growth factor—stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

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Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.955 ◽

1991 ◽

Vol 112 (5) ◽

pp. 955-963 ◽

Cited By ~ 25

Author(s):

P A Maher

Keyword(s):

Embryonic Development ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Protein Tyrosine Phosphorylation ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Low Levels ◽

Protein Tyrosine Kinase Activity

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.

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Signal transduction of the human granulocyte-macrophage colony- stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins

Blood ◽

10.1182/blood.v76.4.706.706 ◽

1990 ◽

Vol 76 (4) ◽

pp. 706-715 ◽

Cited By ~ 161

Author(s):

Y Kanakura ◽

B Druker ◽

SA Cannistra ◽

Y Furukawa ◽

Y Torimoto ◽

...

Keyword(s):

Signal Transduction ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine- kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM- CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM- CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.

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