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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 inhibition of Src kinases on Syk and its substrates. Platelets were processed as described in the legends to Figs. 1 and 5. (A) The effect of 5 μM PP2 on tyrosine phosphorylation of Syk. (B) The effect on tyrosine phosphorylation of Vav1, Vav3 and SLP-76. (C) The effect on adhesion-dependent association of Syk with αIIbβ3. Results are representative of two experiments.
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fig7: Effect of inhibition of Src kinases on Syk and its substrates. Platelets were processed as described in the legends to Figs. 1 and 5. (A) The effect of 5 μM PP2 on tyrosine phosphorylation of Syk. (B) The effect on tyrosine phosphorylation of Vav1, Vav3 and SLP-76. (C) The effect on adhesion-dependent association of Syk with αIIbβ3. Results are representative of two experiments.

Mentions: To identify potential downstream effectors of αIIbβ3 and Src, the effect of PP2 or SU6656 on adhesion-dependent tyrosine phosphorylation of several platelet proteins was examined. The results with PP2 are shown in Fig. 7 and are identical to those obtained with SU6656. PP2, but not PP3, inhibited adhesion-dependent tyrosine phosphorylation of Syk (Fig. 7 A), tyrosine phosphorylation of putative Syk substrates Vav1, Vav3, and SLP-76, and tyrosine phosphorylation of SLAP-130, an adaptor that binds to SLP-76 (Fig. 7 B) (Judd et al., 2000; Obergfell et al., 2001). On the other hand, PP2 had no effect on the adhesion-dependent association of Syk with αIIbβ3 (Fig. 7 C). Thus, one or more Src family kinases appear to be required for αIIbβ3-dependent activation of Syk and for tyrosine phosphorylation of Syk substrates implicated in cytoskeletal regulation (Judd et al., 2000; Obergfell et al., 2001).


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 inhibition of Src kinases on Syk and its substrates. Platelets were processed as described in the legends to Figs. 1 and 5. (A) The effect of 5 μM PP2 on tyrosine phosphorylation of Syk. (B) The effect on tyrosine phosphorylation of Vav1, Vav3 and SLP-76. (C) The effect on adhesion-dependent association of Syk with αIIbβ3. Results are representative of two experiments.
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Related In: Results  -  Collection

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

fig7: Effect of inhibition of Src kinases on Syk and its substrates. Platelets were processed as described in the legends to Figs. 1 and 5. (A) The effect of 5 μM PP2 on tyrosine phosphorylation of Syk. (B) The effect on tyrosine phosphorylation of Vav1, Vav3 and SLP-76. (C) The effect on adhesion-dependent association of Syk with αIIbβ3. Results are representative of two experiments.
Mentions: To identify potential downstream effectors of αIIbβ3 and Src, the effect of PP2 or SU6656 on adhesion-dependent tyrosine phosphorylation of several platelet proteins was examined. The results with PP2 are shown in Fig. 7 and are identical to those obtained with SU6656. PP2, but not PP3, inhibited adhesion-dependent tyrosine phosphorylation of Syk (Fig. 7 A), tyrosine phosphorylation of putative Syk substrates Vav1, Vav3, and SLP-76, and tyrosine phosphorylation of SLAP-130, an adaptor that binds to SLP-76 (Fig. 7 B) (Judd et al., 2000; Obergfell et al., 2001). On the other hand, PP2 had no effect on the adhesion-dependent association of Syk with αIIbβ3 (Fig. 7 C). Thus, one or more Src family kinases appear to be required for αIIbβ3-dependent activation of Syk and for tyrosine phosphorylation of Syk substrates implicated in cytoskeletal regulation (Judd et al., 2000; Obergfell et al., 2001).

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