Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK

1993 ◽  
Vol 13 (2) ◽  
pp. 785-791
Author(s):  
M D Schaller ◽  
C A Borgman ◽  
J T Parsons
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Protein Tyrosine ◽  
Intracellular Signals ◽  
Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

scholarly journals Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK.

1993 ◽  
Vol 13 (2) ◽  
pp. 785-791 ◽  
Author(s):  
M D Schaller ◽  
C A Borgman ◽  
J T Parsons
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Protein Tyrosine ◽  
Intracellular Signals ◽  
Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

scholarly journals Endothelin-1 stimulates tyrosine phosphorylation of p125 focal adhesion kinase in mesangial cells.

1995 ◽  
Vol 6 (5) ◽  
pp. 1504-1510
Author(s):  
M Haneda ◽  
R Kikkawa ◽  
D Koya ◽  
T Shikano ◽  
T Sugimoto ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Protein Tyrosine ◽  
Endothelin 1

Endothelin-1 (ET-1) is known to induce the contraction and proliferation of glomerular mesangial cells. Because ET-1 was found to stimulate the tyrosine phosphorylation of unidentified cellular proteins in cultured mesangial cells, protein tyrosine kinase might serve as one of the important signals leading to various functions of ET-1. Focal adhesion kinase (p125FAK) is a newly identified cytoplasmic protein tyrosine kinase that is activated by the phosphorylation of its own tyrosine residue. Because p125FAK was found to play a role in the signal transduction of not only integrins but also various neurotransmitters, including bombesin, endothelin, and vasopressin in Swiss 3T3 cells and Rat-1 fibroblasts, whether ET-1 could stimulate the tyrosine phosphorylation of p125FAK in glomerular mesangial cells was examined. ET-1 stimulated the tyrosine phosphorylation of p125FAK by threefold to fourfold in cultured mesangial cells. This effect of ET-1 was detected at 1 min and reached a maximum within 5 min and was blocked by BQ-123, an antagonist for ETA receptor. A23187, a calcium ionophore, failed to stimulate the tyrosine phosphorylation of p125FAK, and ET-1 was able to stimulate the tyrosine phosphorylation of p125FAK, even in a calcium-free medium. The activation of protein kinase C (PKC) by phorbol 12, 13-dibutyrate resulted in a stimulation of the tyrosine phosphorylation of p125FAK, and an inhibition of PKC by calphostin C or staurosporine significantly reduced the effect of ET-1. Furthermore, prolonged treatment of the cells with phorbol 12, 13-dibutyrate markedly inhibited the ET-1-induced tyrosine phosphorylation of p125FAK. These results indicate that p125FAK might play a role in a signal transduction system of ET-1 in glomerular mesangial cells and that the ET-1-induced tyrosine phosphorylation of p125FAK is largely dependent on the PKC pathway.

Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin

1996 ◽  
Vol 109 (5) ◽  
pp. 1133-1141 ◽  
Author(s):  
H.M. Flinn ◽  
A.J. Ridley
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Kinase Inhibitors ◽  
Adhesion Formation ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Similar Response ◽  
Rho Proteins ◽  
Swiss 3T3

The small GTP-binding protein Rho rapidly stimulates the formation of focal adhesions and actin stress fibres when microinjected into serum-starved Swiss 3T3 fibroblasts. This response is inhibited by tyrosine kinase inhibitors. Addition of growth factors such as lysophosphatidic acid and bombesin to Swiss 3T3 cells stimulates a similar response, which is dependent on endogenous Rho proteins. To investigate signalling events regulated by Rho, we have scrape loaded Rho into serum-starved cells. Activated Rho stimulates the tyrosine phosphorylation of a number of proteins, including three proteins known to localise to focal adhesions, pp125FAK, p130 and paxillin. Rho-induced phosphorylation of pp125FAK, p130 and paxillin is observed in the absence of stress fibre formation and is, therefore, independent of Rho-induced actin polymerisation. We propose that the tyrosine kinase, pp125FAK, and the putative adapter proteins, paxillin and p130, are components of a Rho-regulated signal transduction pathway, and that these protein tyrosine phosphorylation events are likely to be important for the regulation of focal adhesion formation.

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

1992 ◽  
Vol 119 (4) ◽  
pp. 905-912 ◽  
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.

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

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
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.

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

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.

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.

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

1996 ◽  
Vol 109 (3) ◽  
pp. 699-704 ◽  
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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