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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 platelet adhesion to fibrinogen on Src activation. As described in the legend to Fig. 1, lysates from fibrinogen-adherent and -nonadherent platelets were immunoprecipitated with an antibody to β3 (A) or FAK (B), and immunoprecipitates were probed on Western blots as indicated. (C) An analysis of the pool of Src that did not coimmunoprecipitate with αIIbβ3 after two sequential immunoprecipitations with the anti-β3 antibody. In all panels, platelets were pretreated for 10 min with buffer, cytochalasin D (CD), or diluent (DMSO) before plating. (D) The raw data from the single experiment in A as Src “activity” associated with β3, expressed as the normalized ratio of Src pTyr-418/pTyr-529. (E) The means ± SEM of this ratio for five experiments. (F) The effects of platelet adhesion on Src activity in β3 immunoprecipitates, measured by an in vitro kinase assay as described in Materials and methods. Results represent means ± SEM of four experiments.
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fig2: Effect of platelet adhesion to fibrinogen on Src activation. As described in the legend to Fig. 1, lysates from fibrinogen-adherent and -nonadherent platelets were immunoprecipitated with an antibody to β3 (A) or FAK (B), and immunoprecipitates were probed on Western blots as indicated. (C) An analysis of the pool of Src that did not coimmunoprecipitate with αIIbβ3 after two sequential immunoprecipitations with the anti-β3 antibody. In all panels, platelets were pretreated for 10 min with buffer, cytochalasin D (CD), or diluent (DMSO) before plating. (D) The raw data from the single experiment in A as Src “activity” associated with β3, expressed as the normalized ratio of Src pTyr-418/pTyr-529. (E) The means ± SEM of this ratio for five experiments. (F) The effects of platelet adhesion on Src activity in β3 immunoprecipitates, measured by an in vitro kinase assay as described in Materials and methods. Results represent means ± SEM of four experiments.

Mentions: To begin to explore relationships between αIIbβ3 and Src in outside-in signaling, we asked whether Src is associated with αIIbβ3, either before or after platelet adhesion to fibrinogen, a response dependent on αIIbβ3. Human platelets were incubated for 45 min over a BSA matrix, to which they do not adhere, or a fibrinogen matrix, to which they adhere and gradually spread. Then platelets were solubilized in a buffer containing NP-40 detergent, the αIIbβ3 complex was immunoprecipitated with a polyclonal antibody to β3, and immunoprecipitates were probed on Western blots with an antibody to Src. As shown in Fig. 1 A, Src was detected in β3 immunoprecipitates whether or not the platelets had become adherent to fibrinogen. The same results were obtained if A2A9, an αIIbβ3 complex–dependent antibody, was used to immunoprecipitate the integrin (unpublished data). A comparison of the Src that did and did not quantitatively coprecipitate with αIIbβ3 indicated that ∼3% of the Src solubilized from platelets was associated with αIIbβ3. Fyn and Yes, the only other Src family members tested, were also associated with αIIbβ3 (Fig. 1 A). Note in Fig. 1 A that the amounts of β3 and Src were slightly less in β3 immunoprecipitates from adherent platelets compared with nonadherent ones. This was attributed to a minor redistribution of these proteins to the detergent-insoluble actin cytoskeleton because it was prevented by preincubation of platelets with 10 μM cytochalasin D to inhibit actin polymerization (see Fig. 2 A).


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 platelet adhesion to fibrinogen on Src activation. As described in the legend to Fig. 1, lysates from fibrinogen-adherent and -nonadherent platelets were immunoprecipitated with an antibody to β3 (A) or FAK (B), and immunoprecipitates were probed on Western blots as indicated. (C) An analysis of the pool of Src that did not coimmunoprecipitate with αIIbβ3 after two sequential immunoprecipitations with the anti-β3 antibody. In all panels, platelets were pretreated for 10 min with buffer, cytochalasin D (CD), or diluent (DMSO) before plating. (D) The raw data from the single experiment in A as Src “activity” associated with β3, expressed as the normalized ratio of Src pTyr-418/pTyr-529. (E) The means ± SEM of this ratio for five experiments. (F) The effects of platelet adhesion on Src activity in β3 immunoprecipitates, measured by an in vitro kinase assay as described in Materials and methods. Results represent means ± SEM of four experiments.
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Related In: Results  -  Collection

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fig2: Effect of platelet adhesion to fibrinogen on Src activation. As described in the legend to Fig. 1, lysates from fibrinogen-adherent and -nonadherent platelets were immunoprecipitated with an antibody to β3 (A) or FAK (B), and immunoprecipitates were probed on Western blots as indicated. (C) An analysis of the pool of Src that did not coimmunoprecipitate with αIIbβ3 after two sequential immunoprecipitations with the anti-β3 antibody. In all panels, platelets were pretreated for 10 min with buffer, cytochalasin D (CD), or diluent (DMSO) before plating. (D) The raw data from the single experiment in A as Src “activity” associated with β3, expressed as the normalized ratio of Src pTyr-418/pTyr-529. (E) The means ± SEM of this ratio for five experiments. (F) The effects of platelet adhesion on Src activity in β3 immunoprecipitates, measured by an in vitro kinase assay as described in Materials and methods. Results represent means ± SEM of four experiments.
Mentions: To begin to explore relationships between αIIbβ3 and Src in outside-in signaling, we asked whether Src is associated with αIIbβ3, either before or after platelet adhesion to fibrinogen, a response dependent on αIIbβ3. Human platelets were incubated for 45 min over a BSA matrix, to which they do not adhere, or a fibrinogen matrix, to which they adhere and gradually spread. Then platelets were solubilized in a buffer containing NP-40 detergent, the αIIbβ3 complex was immunoprecipitated with a polyclonal antibody to β3, and immunoprecipitates were probed on Western blots with an antibody to Src. As shown in Fig. 1 A, Src was detected in β3 immunoprecipitates whether or not the platelets had become adherent to fibrinogen. The same results were obtained if A2A9, an αIIbβ3 complex–dependent antibody, was used to immunoprecipitate the integrin (unpublished data). A comparison of the Src that did and did not quantitatively coprecipitate with αIIbβ3 indicated that ∼3% of the Src solubilized from platelets was associated with αIIbβ3. Fyn and Yes, the only other Src family members tested, were also associated with αIIbβ3 (Fig. 1 A). Note in Fig. 1 A that the amounts of β3 and Src were slightly less in β3 immunoprecipitates from adherent platelets compared with nonadherent ones. This was attributed to a minor redistribution of these proteins to the detergent-insoluble actin cytoskeleton because it was prevented by preincubation of platelets with 10 μM cytochalasin D to inhibit actin polymerization (see Fig. 2 A).

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