Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets
Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphoryl...
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2023-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/9/7776 |
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| author | Pengyu Zhang Fiorella A. Solari Johan W. M. Heemskerk Marijke J. E. Kuijpers Albert Sickmann Ulrich Walter Kerstin Jurk |
| author_facet | Pengyu Zhang Fiorella A. Solari Johan W. M. Heemskerk Marijke J. E. Kuijpers Albert Sickmann Ulrich Walter Kerstin Jurk |
| author_sort | Pengyu Zhang |
| collection | DOAJ |
| description | Bruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI–Syk–Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis. |
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| spelling | doaj.art-03c5d5c575c84027bc1a40dfed163e352023-11-17T23:00:29ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-04-01249777610.3390/ijms24097776Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human PlateletsPengyu Zhang0Fiorella A. Solari1Johan W. M. Heemskerk2Marijke J. E. Kuijpers3Albert Sickmann4Ulrich Walter5Kerstin Jurk6Leibniz Institut für Analytische Wissenschaften-ISAS-e.V., 44139 Dortmund, GermanyLeibniz Institut für Analytische Wissenschaften-ISAS-e.V., 44139 Dortmund, GermanyDepartment of Biochemistry, CARIM, Maastricht University, 6229 ER Maastricht, The NetherlandsDepartment of Biochemistry, CARIM, Maastricht University, 6229 ER Maastricht, The NetherlandsLeibniz Institut für Analytische Wissenschaften-ISAS-e.V., 44139 Dortmund, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyBruton’s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI–Syk–Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.https://www.mdpi.com/1422-0067/24/9/7776thrombo-inflammationplateletsprotein kinasesBruton’s tyrosine kinaseXLA |
| spellingShingle | Pengyu Zhang Fiorella A. Solari Johan W. M. Heemskerk Marijke J. E. Kuijpers Albert Sickmann Ulrich Walter Kerstin Jurk Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets International Journal of Molecular Sciences thrombo-inflammation platelets protein kinases Bruton’s tyrosine kinase XLA |
| title | Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets |
| title_full | Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets |
| title_fullStr | Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets |
| title_full_unstemmed | Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets |
| title_short | Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets |
| title_sort | differential regulation of gpvi induced btk and syk activation by pkc pka and pp2a in human platelets |
| topic | thrombo-inflammation platelets protein kinases Bruton’s tyrosine kinase XLA |
| url | https://www.mdpi.com/1422-0067/24/9/7776 |
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