Feedback Regulation of Syk by Protein Kinase C in Human Platelets
The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphor...
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MDPI AG
2019-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/1/176 |
| _version_ | 1811219309460979712 |
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| author | Stephanie Makhoul Stephanie Dorschel Stepan Gambaryan Ulrich Walter Kerstin Jurk |
| author_facet | Stephanie Makhoul Stephanie Dorschel Stepan Gambaryan Ulrich Walter Kerstin Jurk |
| author_sort | Stephanie Makhoul |
| collection | DOAJ |
| description | The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca<sup>2+</sup>. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets. |
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| language | English |
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| spelling | doaj.art-bc81a9c0077642578baaf3a88f75b8e72022-12-22T03:42:14ZengMDPI AGInternational Journal of Molecular Sciences1422-00672019-12-0121117610.3390/ijms21010176ijms21010176Feedback Regulation of Syk by Protein Kinase C in Human PlateletsStephanie Makhoul0Stephanie Dorschel1Stepan Gambaryan2Ulrich Walter3Kerstin Jurk4Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center Mainz of the Johannes Gutenberg University Mainz, 55131 Mainz, GermanyThe spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca<sup>2+</sup>. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.https://www.mdpi.com/1422-0067/21/1/176spleen tyrosine kinase (syk)protein kinase ccyclic adenosine monophosphate (camp)plateletsglycoprotein viglycoprotein ibα |
| spellingShingle | Stephanie Makhoul Stephanie Dorschel Stepan Gambaryan Ulrich Walter Kerstin Jurk Feedback Regulation of Syk by Protein Kinase C in Human Platelets International Journal of Molecular Sciences spleen tyrosine kinase (syk) protein kinase c cyclic adenosine monophosphate (camp) platelets glycoprotein vi glycoprotein ibα |
| title | Feedback Regulation of Syk by Protein Kinase C in Human Platelets |
| title_full | Feedback Regulation of Syk by Protein Kinase C in Human Platelets |
| title_fullStr | Feedback Regulation of Syk by Protein Kinase C in Human Platelets |
| title_full_unstemmed | Feedback Regulation of Syk by Protein Kinase C in Human Platelets |
| title_short | Feedback Regulation of Syk by Protein Kinase C in Human Platelets |
| title_sort | feedback regulation of syk by protein kinase c in human platelets |
| topic | spleen tyrosine kinase (syk) protein kinase c cyclic adenosine monophosphate (camp) platelets glycoprotein vi glycoprotein ibα |
| url | https://www.mdpi.com/1422-0067/21/1/176 |
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