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Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton.

Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ - J. Cell Biol. (2002)

Bottom Line: Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges.Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3.In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.

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Effect of soluble fibrinogen binding to platelets on αIIbβ3-associated Csk and Src. Platelets were incubated as indicated in the presence or absence of 250 μg/ml fibrinogen, 0.5 mM MnCl2, and 2 mM RGDS for 20 min. Then the presence of Csk and Src in β3 immunoprecipitates was analyzed as described in the legends to Figs. 1 and 2. Results are representative of two experiments.
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fig3: Effect of soluble fibrinogen binding to platelets on αIIbβ3-associated Csk and Src. Platelets were incubated as indicated in the presence or absence of 250 μg/ml fibrinogen, 0.5 mM MnCl2, and 2 mM RGDS for 20 min. Then the presence of Csk and Src in β3 immunoprecipitates was analyzed as described in the legends to Figs. 1 and 2. Results are representative of two experiments.

Mentions: In contrast to platelet adhesion to immobilized fibrinogen, binding of soluble fibrinogen to platelets requires prior activation of αIIbβ3. Therefore, to investigate whether soluble fibrinogen binding is sufficient to activate Src, platelets were incubated in the presence of 250 μg/ml fibrinogen, and 0.5 mM MnCl2 was added to directly activate αIIbβ3 (Bazzoni and Hemler, 1998). Fibrinogen binding caused both the dissociation of Csk from the αIIbβ3 complex and the increased phosphorylation of Src Tyr-418 (Fig. 3, Fib + Mn2+ lane). These responses were observed as early as 1 min, were stable for at least 20 min, and were blocked by 2 mM RGDS, which inhibits fibrinogen binding to αIIbβ3. Interestingly, MnCl2 or RGDS each induced a small amount Csk dissociation and Src Tyr-418 phosphorylation, suggesting that both integrin activation and ligation contribute to Src activation (Fig. 3). Together with the data for adherent platelets, these results establish that fibrinogen binding to αIIbβ3 causes dissociation of Csk from the αIIbβ3 complex at the same time that the integrin-associated pool of Src becomes activated. These responses require neither actin polymerization nor tyrosine phosphorylation of FAK.


Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton.

Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ - J. Cell Biol. (2002)

Effect of soluble fibrinogen binding to platelets on αIIbβ3-associated Csk and Src. Platelets were incubated as indicated in the presence or absence of 250 μg/ml fibrinogen, 0.5 mM MnCl2, and 2 mM RGDS for 20 min. Then the presence of Csk and Src in β3 immunoprecipitates was analyzed as described in the legends to Figs. 1 and 2. Results are representative of two experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199242&req=5

fig3: Effect of soluble fibrinogen binding to platelets on αIIbβ3-associated Csk and Src. Platelets were incubated as indicated in the presence or absence of 250 μg/ml fibrinogen, 0.5 mM MnCl2, and 2 mM RGDS for 20 min. Then the presence of Csk and Src in β3 immunoprecipitates was analyzed as described in the legends to Figs. 1 and 2. Results are representative of two experiments.
Mentions: In contrast to platelet adhesion to immobilized fibrinogen, binding of soluble fibrinogen to platelets requires prior activation of αIIbβ3. Therefore, to investigate whether soluble fibrinogen binding is sufficient to activate Src, platelets were incubated in the presence of 250 μg/ml fibrinogen, and 0.5 mM MnCl2 was added to directly activate αIIbβ3 (Bazzoni and Hemler, 1998). Fibrinogen binding caused both the dissociation of Csk from the αIIbβ3 complex and the increased phosphorylation of Src Tyr-418 (Fig. 3, Fib + Mn2+ lane). These responses were observed as early as 1 min, were stable for at least 20 min, and were blocked by 2 mM RGDS, which inhibits fibrinogen binding to αIIbβ3. Interestingly, MnCl2 or RGDS each induced a small amount Csk dissociation and Src Tyr-418 phosphorylation, suggesting that both integrin activation and ligation contribute to Src activation (Fig. 3). Together with the data for adherent platelets, these results establish that fibrinogen binding to αIIbβ3 causes dissociation of Csk from the αIIbβ3 complex at the same time that the integrin-associated pool of Src becomes activated. These responses require neither actin polymerization nor tyrosine phosphorylation of FAK.

Bottom Line: Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges.Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3.In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding.

View Article: PubMed Central - PubMed

Affiliation: Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.

Show MeSH
Related in: MedlinePlus