scholarly journals Collagen But Not Fibrinogen Surfaces Induce Bleb Formation, Exposure of Phosphatidylserine, and Procoagulant Activity of Adherent Platelets: Evidence for Regulation by Protein Tyrosine Kinase-Dependent Ca2+ Responses

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2615-2625 ◽  
Author(s):  
Johan W.M. Heemskerk ◽  
Wim M.J. Vuist ◽  
Marion A.H. Feijge ◽  
Chris P.M. Reutelingsperger ◽  
Theo Lindhout
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Imaging System ◽  
Morphological Changes ◽  
Annexin V ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Protein Tyrosine ◽  
The Matrix ◽  
Bleb Formation

With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

scholarly journals Collagen But Not Fibrinogen Surfaces Induce Bleb Formation, Exposure of Phosphatidylserine, and Procoagulant Activity of Adherent Platelets: Evidence for Regulation by Protein Tyrosine Kinase-Dependent Ca2+ Responses

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2615-2625 ◽  
Author(s):  
Johan W.M. Heemskerk ◽  
Wim M.J. Vuist ◽  
Marion A.H. Feijge ◽  
Chris P.M. Reutelingsperger ◽  
Theo Lindhout
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Imaging System ◽  
Morphological Changes ◽  
Annexin V ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Protein Tyrosine ◽  
The Matrix ◽  
Bleb Formation

Abstract With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

scholarly journals Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 240
Author(s):  
Lan-Yi Wei ◽  
Wei Lin ◽  
Bey-Fen Leo ◽  
Lik-Voon Kiew ◽  
Chia-Ching Chang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Significant Inhibition ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Sensing Platform ◽  
Relative Standard ◽  
Protein Tyrosine Kinase Activity

A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 μL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases.

Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning

2003 ◽  
Vol 284 (4) ◽  
pp. H1441-H1448 ◽  
Author(s):  
Xian-Liang Tang ◽  
Eitaro Kodani ◽  
Hitoshi Takano ◽  
Michael Hill ◽  
Ken Shinmura ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Myocardial Stunning ◽  
Protein Tyrosine Kinase ◽  
Late Phase ◽  
Recovery Period ◽  
Wall Thickening ◽  
No Donor ◽  
Signaling Mechanism ◽  
Protein Tyrosine ◽  
Conscious Rabbits

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days ( days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits ( n = 6), the six O/R cycles on day 1resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO ( n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day 1 was markedly attenuated, with a significant reduction (∼60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 ( n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 ( n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.

scholarly journals Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

1995 ◽  
Vol 130 (5) ◽  
pp. 1181-1187 ◽  
Author(s):  
M D Schaller ◽  
C A Otey ◽  
J D Hildebrand ◽  
J T Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Embryo Cell ◽  
Vitro Assay ◽  
Protein Tyrosine ◽  
Signaling Complex

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps

1989 ◽  
Vol 9 (5) ◽  
pp. 2214-2219
Author(s):  
A R Brooks-Wilson ◽  
E Ball ◽  
T Pawson
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Specific Region ◽  
Protein Tyrosine Kinases ◽  
Membrane Association ◽  
Modular Construction ◽  
Specific Domain ◽  
Protein Tyrosine ◽  
Sarcoma Virus ◽  
Transforming Activity

The P130gag-fps protein-tyrosine kinase of Fujinami sarcoma virus contains an N-terminal fps-specific domain (Nfps) that is important for oncogenicity. The N-terminal 14 amino acids of p60v-src, which direct myristylation and membrane association, can replace the gag-Nfps sequences of P130gag-fps (residues 1 to 635), producing a highly transforming src-fps polypeptide. Conversely, gag-Nfps can restore modest transforming activity to a nonmyristylated v-src polypeptide. These results emphasize the modular construction of protein-tyrosine kinases and indicate that Nfps, possibly in conjunction with gag, functions in the subcellular localization of P130gag-fps.

Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene

1994 ◽  
Vol 14 (9) ◽  
pp. 5812-5819
Author(s):  
H Shibuya ◽  
K Kohu ◽  
K Yamada ◽  
E L Barsoumian ◽  
R M Perlmutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Gene Activation ◽  
Interleukin 2 ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Amino Acid Residues ◽  
Protein Tyrosine ◽  
Responsive Element

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK

1994 ◽  
Vol 14 (1) ◽  
pp. 147-155
Author(s):  
B S Cobb ◽  
M D Schaller ◽  
T H Leu ◽  
J T Parsons
Keyword(s):  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Src Family Kinases ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Sh2 Domains ◽  
Embryo Cells ◽  
Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Characterization of elk, a brain-specific receptor tyrosine kinase

1991 ◽  
Vol 11 (5) ◽  
pp. 2496-2502
Author(s):  
V Lhoták ◽  
P Greer ◽  
K Letwin ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Catalytic Domain ◽  
Signal Sequence ◽  
Tyrosine Kinase Domain ◽  
Rat Tissues ◽  
Protein Tyrosine

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

scholarly journals Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

1994 ◽  
Vol 14 (7) ◽  
pp. 4509-4521 ◽  
Author(s):  
Z Weng ◽  
S M Thomas ◽  
R J Rickles ◽  
J A Taylor ◽  
A W Brauer ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Binding Proteins ◽  
Direct Interaction ◽  
Sh3 Domain ◽  
Peptide Ligand ◽  
Sh3 Domains ◽  
Protein Tyrosine ◽  
Peptide Motifs

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

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