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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 Syk deficiency on adhesion-dependent tyrosine phosphorylation in platelets. After plating on BSA or fibrinogen for 45 min, wild-type and syk−/− platelets were subjected to immunoprecipitation and Western blotting as indicated. Results are representative of three experiments.
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fig9: Effect of Syk deficiency on adhesion-dependent tyrosine phosphorylation in platelets. After plating on BSA or fibrinogen for 45 min, wild-type and syk−/− platelets were subjected to immunoprecipitation and Western blotting as indicated. Results are representative of three experiments.

Mentions: To identify platelet proteins whose tyrosine phosphorylation is dependent on Syk, syk−/− platelets were subjected to immunoprecipitation and Western blot analysis. Compared with wild-type platelets, syk−/− platelets exhibited virtually no adhesion-dependent tyrosine phosphorylation of Vav1, Vav3, or SLP-76, and reduced tyrosine phosphorylation of SLAP-130 (Fig. 9). In contrast, both the association of Src with αIIbβ3 and the adhesion-dependent activation of Src were normal in syk−/− platelets (Fig. 10). Altogether, these results indicate that Syk is downstream of Src but upstream of Vav1, Vav3, and SLP-76 in an αIIbβ3 signaling pathway that regulates the platelet actin cytoskeleton.


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 Syk deficiency on adhesion-dependent tyrosine phosphorylation in platelets. After plating on BSA or fibrinogen for 45 min, wild-type and syk−/− platelets were subjected to immunoprecipitation and Western blotting as indicated. Results are representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Effect of Syk deficiency on adhesion-dependent tyrosine phosphorylation in platelets. After plating on BSA or fibrinogen for 45 min, wild-type and syk−/− platelets were subjected to immunoprecipitation and Western blotting as indicated. Results are representative of three experiments.
Mentions: To identify platelet proteins whose tyrosine phosphorylation is dependent on Syk, syk−/− platelets were subjected to immunoprecipitation and Western blot analysis. Compared with wild-type platelets, syk−/− platelets exhibited virtually no adhesion-dependent tyrosine phosphorylation of Vav1, Vav3, or SLP-76, and reduced tyrosine phosphorylation of SLAP-130 (Fig. 9). In contrast, both the association of Src with αIIbβ3 and the adhesion-dependent activation of Src were normal in syk−/− platelets (Fig. 10). Altogether, these results indicate that Syk is downstream of Src but upstream of Vav1, Vav3, and SLP-76 in an αIIbβ3 signaling pathway that regulates the platelet actin cytoskeleton.

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