Limits...
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.

Show MeSH

Related in: MedlinePlus

Platelet spreading on fibrinogen is defective in syk−/− murine platelets. Platelets were obtained from syk−/− and wild-type mice and plated on fibrinogen for 45 min with no agonist; with a combination of PAR-4 receptor–activating peptide (1 mM), ADP (50 μM), and epinephrine (50 μM); or with collagen (10 μg/ml). Cells were then fixed, permeabilized and stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads point to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199242&req=5

fig8: Platelet spreading on fibrinogen is defective in syk−/− murine platelets. Platelets were obtained from syk−/− and wild-type mice and plated on fibrinogen for 45 min with no agonist; with a combination of PAR-4 receptor–activating peptide (1 mM), ADP (50 μM), and epinephrine (50 μM); or with collagen (10 μg/ml). Cells were then fixed, permeabilized and stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads point to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. Bar, 10 μm.

Mentions: Platelets genetically deficient in Syk were evaluated to establish if Syk was required for either Src activation or for cytoskeletal changes that promote platelet spreading. The perinatal lethality of syk−/− mice was overcome by generation of syk−/− bone marrow chimeras. Compared with wild-type platelets, syk−/− platelets displayed fewer and less prominent filopodia, reduced tyrosine phosphorylation, and less spreading and (P < 0.001) (Fig. 8). However, when syk−/− platelets were stimulated during the adhesion process with a combination of PAR-4 receptor–activating peptide, ADP, and epinephrine, they now spread fully and exhibited tyrosine phosphorylation at cell edges (Fig. 8). As expected, syk−/− platelets spread poorly on fibrinogen when stimulated with collagen, which activates platelets in part through an Syk-coupled receptor, GP VI/FcR γ (Watson and Gibbins, 1998). Thus, Syk is required for platelet spreading mediated by outside-in signaling through αIIbβ3. However, spreading can occur in a manner independent of Syk if platelets are costimulated with agonists to G protein–coupled receptors.


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)

Platelet spreading on fibrinogen is defective in syk−/− murine platelets. Platelets were obtained from syk−/− and wild-type mice and plated on fibrinogen for 45 min with no agonist; with a combination of PAR-4 receptor–activating peptide (1 mM), ADP (50 μM), and epinephrine (50 μM); or with collagen (10 μg/ml). Cells were then fixed, permeabilized and stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads point to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Platelet spreading on fibrinogen is defective in syk−/− murine platelets. Platelets were obtained from syk−/− and wild-type mice and plated on fibrinogen for 45 min with no agonist; with a combination of PAR-4 receptor–activating peptide (1 mM), ADP (50 μM), and epinephrine (50 μM); or with collagen (10 μg/ml). Cells were then fixed, permeabilized and stained for F-actin (red) and phosphotyrosine (green), and analyzed by confocal microscopy. Arrowheads point to filopodia and arrows to the peripheral rims of some of the spreading platelets. Results are representative of three experiments. Bar, 10 μm.
Mentions: Platelets genetically deficient in Syk were evaluated to establish if Syk was required for either Src activation or for cytoskeletal changes that promote platelet spreading. The perinatal lethality of syk−/− mice was overcome by generation of syk−/− bone marrow chimeras. Compared with wild-type platelets, syk−/− platelets displayed fewer and less prominent filopodia, reduced tyrosine phosphorylation, and less spreading and (P < 0.001) (Fig. 8). However, when syk−/− platelets were stimulated during the adhesion process with a combination of PAR-4 receptor–activating peptide, ADP, and epinephrine, they now spread fully and exhibited tyrosine phosphorylation at cell edges (Fig. 8). As expected, syk−/− platelets spread poorly on fibrinogen when stimulated with collagen, which activates platelets in part through an Syk-coupled receptor, GP VI/FcR γ (Watson and Gibbins, 1998). Thus, Syk is required for platelet spreading mediated by outside-in signaling through αIIbβ3. However, spreading can occur in a manner independent of Syk if platelets are costimulated with agonists to G protein–coupled receptors.

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