In Vitro Assays for the Detection of Protein Tyrosine Phosphorylation and Protein Tyrosine Kinase Activities

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-α-stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-α-stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 μM), but not genistin, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-α-stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-α upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

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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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SRC family kinases in hamster spermatozoa: evidence for the presence of LCK

Reproduction ◽

10.1530/rep-16-0591 ◽

2017 ◽

Vol 153 (5) ◽

pp. 655-669 ◽

Cited By ~ 3

Author(s):

Durgesh Kumar Singh ◽

Rohit Kumar Deshmukh ◽

Praveen Kumar Narayanan ◽

Sisinthy Shivaji ◽

Archana Bharadwaj Siva

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Specific Inhibitor ◽

Specific Protein ◽

Src Family Kinases ◽

Sperm Capacitation ◽

Protein Tyrosine

Sperm capacitation is a prerequisite for successful fertilization. Increase in tyrosine phosphorylation is considered the hallmark of capacitation and attempts to understand its regulation are ongoing. In this regard, we attempted to study the role of SRC family kinases (SFKs) in the hamster sperm functions. Interestingly, we found the presence of the lymphocyte-specific protein tyrosine kinase, LCK, in mammalian spermatozoa and further characterized it in terms of its localization and function. LCK was found in spermatozoa of several species, and its transcript was identified in the hamster testis. Autophosphorylation of LCK at the Y394 residue increased as capacitation progressed, indicating an upregulation of LCK activity during capacitation. Inhibition of LCK (and perhaps the other SFKs) with the use of a specific inhibitor showed a significant decrease in protein tyrosine phosphorylation of several proteins, implying LCK/SFKs as key tyrosine kinase(s) regulating tyrosine phosphorylation during hamster sperm capacitation. Dihydrolipoamide dehydrogenase was identified as a substrate for LCK/SFK. LCK/SFKs inhibition significantly reduced the percentage fertilization (in vitro) but had no effect on sperm motility, hyperactivation and acrosome reaction. In summary, this is the first report on the presence of LCK, an SFK of hematopoietic lineage in spermatozoa besides being the first study on the role of SFKs in the spermatozoa of Syrian hamsters.

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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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Cell proliferation status, cytokine action and protein tyrosine phosphorylation modulate leukotriene biosynthesis in a basophil leukaemia and a mastocytoma cell line

Biochemical Journal ◽

10.1042/bj2990467 ◽

1994 ◽

Vol 299 (2) ◽

pp. 467-472 ◽

Cited By ~ 12

Author(s):

W Hagmann

Keyword(s):

Growth Factors ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitors ◽

Interleukin 4 ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Herbimycin A ◽

Mastocytoma Cells

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

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Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata

Parasitology ◽

10.1017/s0031182007003964 ◽

2007 ◽

Vol 135 (3) ◽

pp. 337-345 ◽

Cited By ~ 7

Author(s):

A. J. WALKER ◽

D. ROLLINSON

Keyword(s):

Tyrosine Kinase ◽

Schistosoma Mansoni ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Fold Increase ◽

Biomphalaria Glabrata ◽

Snail Host ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

SUMMARYMolecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

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Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.

The Journal of Cell Biology ◽

10.1083/jcb.119.4.905 ◽

1992 ◽

Vol 119 (4) ◽

pp. 905-912 ◽

Cited By ~ 457

Author(s):

L Lipfert ◽

B Haimovich ◽

M D Schaller ◽

B S Cobb ◽

J T Parsons ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Immune Complex ◽

Strong Correlation ◽

Actin Polymerization ◽

Protein Tyrosine Kinase ◽

Focal Adhesions ◽

Protein Tyrosine ◽

Fibrinogen Binding

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.

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Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l217 ◽

1997 ◽

Vol 273 (1) ◽

pp. L217-L226 ◽

Cited By ~ 10

Author(s):

D. D. Bannerman ◽

S. E. Goldblum

Keyword(s):

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Endothelial Barrier ◽

Protein Tyrosine Phosphorylation ◽

Gap Formation ◽

Barrier Dysfunction ◽

Protein Tyrosine ◽

Actin Depolymerization ◽

Endothelial Barrier Dysfunction

Bacterial lipopolysaccharide (LPS) induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in vitro. We studied whether LPS-induced increments in 14C-labeled bovine serum albumin (BSA) flux across bovine pulmonary artery endothelial cell (EC) monolayers and actin depolymerization are mediated through protein tyrosine phosphorylation. Lysates from EC exposed to LPS derived from Escherichia coli 0111:B4 (100 ng/ml, 1 h) demonstrated increased tyrosine phosphorylation of the cytoskeletal protein paxillin. Protein tyrosine kinase inhibition, with either herbimycin A (1 microM) or genistein (50 micrograms/ml), protected against LPS-induced actin depolymerization, intercellular gap formation, and increments in [14C]BSA flux. In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate (2.5 microM) or phenylarsine oxide (0.1 microM) enhanced the LPS-induced increments in the G-actin pool and the transendothelial flux of [14C]BSA compared with that seen after exposure to LPS alone. Our data indicate that the influence of LPS on EC actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation.

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Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648987 ◽

1994 ◽

Vol 72 (06) ◽

pp. 937-941 ◽

Cited By ~ 7

Author(s):

Karim Rezaul ◽

Shigeru Yanagi ◽

Kiyonao Sada ◽

Takanobu Taniguchi ◽

Hirohei Yamamura

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Critical Role ◽

Platelet Activating Factor ◽

Kinase Assay ◽

Potential Candidate ◽

Protein Tyrosine ◽

Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

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