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Cited by 40Platelet Disorders and TreatmentsProtein Kinase Regulation and GTPase SignalingCell Adhesion Molecules Research

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The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets

Canobbio, Ilaria, Cipolla, Lina, Guidetti, Gianni F., Manganaro, Daria, Visconte, Caterina, Kim, Soochong, Okigaki, Mitsuhiko, Falasca, Marco, Kunapuli, Satya P., Torti, Mauro·2015·Biochemical Journal
DOI 10.1042/bj20150048
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Abstract

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca2+ and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca2+ and accordingly the Ca2+ ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca2+-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca2+-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2 in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca2+ initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

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Metadata

Authors
Canobbio, I., Cipolla, L., Guidetti, G. F., Manganaro, D., Visconte, C., Kim, S., Okigaki, M., Falasca, M., Kunapuli, S. P., & Torti, M.
Affiliations
  • University of Pavia (IT)
  • Chungbuk National University (KR)
  • Kyoto Prefectural University (JP)
  • Curtin University (AU)
  • Temple University (US)
Corresponding author
  • Canobbio, Ilaria
  • Cipolla, Lina
  • Manganaro, Daria
  • Visconte, Caterina
  • Torti, Mauro
ORCID
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
2015-07-06
Volume
469
Issue
2
Pages
199-210
ISSN
  • 0264-6021 (ISSN-L)
  • 1470-8728
DOI
10.1042/bj20150048
OpenAlex
W1952327286
Cited by
40
References
1
Citations by year
2026: 3 · 2025: 2 · 2023: 2 · 2022: 4 · 2021: 5 · 2020: 7 · 2019: 3 · 2018: 6 · 2017: 4 · 2016: 4
FWCI
3.67
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemical Journal (publishedVersion)
  • PubMed (publishedVersion)
  • eSpace (Curtin University) (submittedVersion)
Funders
National Heart, Lung, and Blood Institute (R01HL118593, R01HL093231, HL118593, HL93231)
Topics
  • Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
Keywords
Pyk2, focal adhesion kinase, Src family kinases, thrombin, platelets, intracellular calcium signaling, tyrosine phosphorylation, Fyn, Lyn kinase, platelet aggregation, G protein-coupled receptor signaling, PP2
MeSH terms
Animals, Blood Platelets, Enzyme Activation, Hemostatics, Humans, Phosphorylation, Thrombin, Platelet Activation, Mice, Knockout, src-Family Kinases, Calcium Signaling, Proto-Oncogene Proteins c-fyn, Mice, Focal Adhesion Kinase 2

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CoChat. (2026). Citation check: The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets. https://app.cochat.ai/papers/10.1042/bj20150048
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Canobbio, I., Cipolla, L., Guidetti, G. F., Manganaro, D., Visconte, C., Kim, S., Okigaki, M., Falasca, M., Kunapuli, S. P., & Torti, M. (2015). The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets. Biochemical Journal. https://doi.org/10.1042/bj20150048