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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 PP2 on Src activity and Src association with αIIbβ3. Platelets were preincubated for 30 min with PP2, plated on fibrinogen or suspended over BSA for 45 min, and Src in αIIbβ3 immunoprecipitates was analyzed as described in the legend to Fig. 1. Results are representative of two experiments.
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fig5: Effect of PP2 on Src activity and Src association with αIIbβ3. Platelets were preincubated for 30 min with PP2, plated on fibrinogen or suspended over BSA for 45 min, and Src in αIIbβ3 immunoprecipitates was analyzed as described in the legend to Fig. 1. Results are representative of two experiments.

Mentions: To determine whether an Src kinase is required for αIIbβ3-dependent cytoskeletal changes, platelets were preincubated for 30 min with PP2, an inhibitor of Src family kinases (Hanke et al., 1996), and then plated on fibrinogen. PP3, an inactive analogue of PP2, was used as a control. 5 μM PP2 abolished adhesion-dependent tyrosine phosphorylation of Src Tyr-418 in β3 immunoprecipitates, but not phosphorylation of Tyr-529 or the association of Src with αIIbβ3 (Fig. 5). Control platelets adherent to fibrinogen underwent morphological changes ranging from filopodia protrusion to complete spreading, with F-actin and tyrosine-phosphorylated proteins evident at the cell periphery. In contrast, platelets treated with 5 μM PP2 generally showed fewer and smaller filopodia and spread very poorly (Fig. 6 A). The differences in spreading between PP2-treated and control platelets were significant as assessed by computerized image analysis of cell surface areas (P < 0.001). Although not shown, results identical to those with PP2 were obtained with 2 μM SU6656, a pharmacologically distinct, selective inhibitor of Src family kinases (Blake et al., 2000). Furthermore, the spreading defect of platelets treated with PP2 or SU6656 could be overcome if the cells were stimulated during adhesion with a combination of agonists to G protein–coupled receptors (1 mM PAR-4 receptor–activating peptide, 50 μM ADP, and 50 μM epinephrine).


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 PP2 on Src activity and Src association with αIIbβ3. Platelets were preincubated for 30 min with PP2, plated on fibrinogen or suspended over BSA for 45 min, and Src in αIIbβ3 immunoprecipitates was analyzed as described in the legend to Fig. 1. Results are representative of two experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effect of PP2 on Src activity and Src association with αIIbβ3. Platelets were preincubated for 30 min with PP2, plated on fibrinogen or suspended over BSA for 45 min, and Src in αIIbβ3 immunoprecipitates was analyzed as described in the legend to Fig. 1. Results are representative of two experiments.
Mentions: To determine whether an Src kinase is required for αIIbβ3-dependent cytoskeletal changes, platelets were preincubated for 30 min with PP2, an inhibitor of Src family kinases (Hanke et al., 1996), and then plated on fibrinogen. PP3, an inactive analogue of PP2, was used as a control. 5 μM PP2 abolished adhesion-dependent tyrosine phosphorylation of Src Tyr-418 in β3 immunoprecipitates, but not phosphorylation of Tyr-529 or the association of Src with αIIbβ3 (Fig. 5). Control platelets adherent to fibrinogen underwent morphological changes ranging from filopodia protrusion to complete spreading, with F-actin and tyrosine-phosphorylated proteins evident at the cell periphery. In contrast, platelets treated with 5 μM PP2 generally showed fewer and smaller filopodia and spread very poorly (Fig. 6 A). The differences in spreading between PP2-treated and control platelets were significant as assessed by computerized image analysis of cell surface areas (P < 0.001). Although not shown, results identical to those with PP2 were obtained with 2 μM SU6656, a pharmacologically distinct, selective inhibitor of Src family kinases (Blake et al., 2000). Furthermore, the spreading defect of platelets treated with PP2 or SU6656 could be overcome if the cells were stimulated during adhesion with a combination of agonists to G protein–coupled receptors (1 mM PAR-4 receptor–activating peptide, 50 μM ADP, and 50 μM epinephrine).

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