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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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Distribution of activated Src and total Src in fibrinogen-adherent platelets. Cells were plated on fibrinogen-coated coverslips for 45 min and prepared for confocal microscopy as described in Materials and methods. Images represent four platelets in various stages of spreading. In the merged images, activated Src (Src pTyr-418) is red and total Src is green. Arrowheads point to some of the filopodia that stained heavily for activated Src. The results are from a single experiment representative of three so performed. Bar, 10 μm.
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fig4: Distribution of activated Src and total Src in fibrinogen-adherent platelets. Cells were plated on fibrinogen-coated coverslips for 45 min and prepared for confocal microscopy as described in Materials and methods. Images represent four platelets in various stages of spreading. In the merged images, activated Src (Src pTyr-418) is red and total Src is green. Arrowheads point to some of the filopodia that stained heavily for activated Src. The results are from a single experiment representative of three so performed. Bar, 10 μm.

Mentions: The results presented so far are consistent with the idea that Src activation may occur in localized regions of the platelet where αIIbβ3 first comes in contact with fibrinogen. Therefore, the distribution of activated Src in fibrinogen-adherent platelets was determined by confocal microscopy using the anti–pTyr-418 antibody as a marker. Although this antibody may also react with the corresponding activation loop phosphotyrosine of other Src family members, it reacted on Western blots of platelet lysates with an apparent single band at ∼60 kD, consistent with Src (unpublished data). Anti–pTyr-418 antibody staining was confined to the filopodia and edges of spreading platelets and to a central region corresponding to the granulomere. In contrast, the distribution of “total” Src determined with antibody 327 was more diffuse (Fig. 4). Thus, Src activation takes place in association with αIIbβ3 and at the periphery of spreading platelets.


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)

Distribution of activated Src and total Src in fibrinogen-adherent platelets. Cells were plated on fibrinogen-coated coverslips for 45 min and prepared for confocal microscopy as described in Materials and methods. Images represent four platelets in various stages of spreading. In the merged images, activated Src (Src pTyr-418) is red and total Src is green. Arrowheads point to some of the filopodia that stained heavily for activated Src. The results are from a single experiment representative of three so performed. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Distribution of activated Src and total Src in fibrinogen-adherent platelets. Cells were plated on fibrinogen-coated coverslips for 45 min and prepared for confocal microscopy as described in Materials and methods. Images represent four platelets in various stages of spreading. In the merged images, activated Src (Src pTyr-418) is red and total Src is green. Arrowheads point to some of the filopodia that stained heavily for activated Src. The results are from a single experiment representative of three so performed. Bar, 10 μm.
Mentions: The results presented so far are consistent with the idea that Src activation may occur in localized regions of the platelet where αIIbβ3 first comes in contact with fibrinogen. Therefore, the distribution of activated Src in fibrinogen-adherent platelets was determined by confocal microscopy using the anti–pTyr-418 antibody as a marker. Although this antibody may also react with the corresponding activation loop phosphotyrosine of other Src family members, it reacted on Western blots of platelet lysates with an apparent single band at ∼60 kD, consistent with Src (unpublished data). Anti–pTyr-418 antibody staining was confined to the filopodia and edges of spreading platelets and to a central region corresponding to the granulomere. In contrast, the distribution of “total” Src determined with antibody 327 was more diffuse (Fig. 4). Thus, Src activation takes place in association with αIIbβ3 and at the periphery of spreading platelets.

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