Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol; Manne, Bhanu Kanth; Badolia, Rachit; Borgognone, Alessandra; Tsygankov, Alexander Y.; McKenzie, Steven E.; Kunapuli, Satya P.

doi:10.1074/jbc.m113.464107

Cited by 15 publications

(14 citation statements)

References 35 publications

(20 reference statements)

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“…Considering the dynamic aspects of Syk activity by phosphorylation/dephosphorylation observed in other cells [22], the effects observed here are substantial. Interestingly, the PKC inhibitor GFX was reported to enhance GPVI-mediated Syk phosphorylation at Y525/526 without affecting the Y352 and Y323 sites in human platelets, effects not observed with murine platelets [46]. However, direct PKC substrates were not investigated.…”

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confidence: 99%

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Makhoul

Dorschel

Gambaryan

et al. 2019

IJMS

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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/Ca2+. 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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confidence: 99%

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Makhoul

Dorschel

Gambaryan

et al. 2019

IJMS

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“…TULA-2 KO mice have been reported to have hyperphosphorylation of Syk at several sites, including Y323 and Y525/526 tyrosine residues. 18,39 In this study we used Y525/526 and Y323 as the readout for Syk activation in HEL cells for platelet FcgRIIA signaling. Crosslinking FcgRIIA by an anti-FcgRIIA antibody (IV.3) and GAM induces receptor clustering and activation, which leads to phosphorylation of Syk in HEL cells.…”

Section: Org Frommentioning

confidence: 99%

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

Zhou

Abraham

André

et al. 2015

Blood

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“…5). In contrast, inhibitors preferentially targeting PKC␤ (e.g., 539654 and Ly 3176155), at concentrations that were inhibitory in other systems (7,8), had little effect on [Ca 2ϩ ] e -evoked [Ca 2ϩ ] i oscillations (Fig. 5.…”

Section: Broad-spectrum Pkc Inhibitors Eliminate [Ca 2ϩ ] E -Evoked [mentioning

confidence: 86%

Intracellular Ca²⁺oscillations generated via the Ca²⁺-sensing receptor are mediated by negative feedback by PKCα at Thr⁸⁸⁸

Young

Rey

Sinnett‐Smith

2014

American Journal of Physiology-Cell Physiology

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To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 μM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).

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Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Cited by 15 publications

References 35 publications

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

Intracellular Ca²⁺oscillations generated via the Ca²⁺-sensing receptor are mediated by negative feedback by PKCα at Thr⁸⁸⁸

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Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Cited by 15 publications

References 35 publications

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

Intracellular Ca2+oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

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Intracellular Ca²⁺oscillations generated via the Ca²⁺-sensing receptor are mediated by negative feedback by PKCα at Thr⁸⁸⁸