A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

Ren, Jianjun; Cook, Aaron A.; Bergmeier, Wolfgang; Sondek, John

doi:10.1016/j.bbrc.2016.04.100

Cited by 11 publications

(10 citation statements)

References 29 publications

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“…This could be explained by the negative regulation of CalDAG-GEFI activity by ERKs, as previously described. 40 All these results support the notion that Shp2 is the phosphatase regulated by ERKs that dephosphorylates and inhibits C3G.…”

Section: Shp2 Colocalizes With C3g and Inhibits Its Gef Activitysupporting

confidence: 72%

“…Taken together, these results suggest the existence of a positive regulatory mechanism mediated by ERKs that, through the inhibition of Shp2, enhances the phosphorylation of C3G induced by thrombin. Interestingly, ERKs inhibit CalDAG-GEFI, 40 which raises the idea that ERKs can positively or negatively regulate the activity of Rap1b through the modulation of its GEFs.…”

Section: Discussionmentioning

confidence: 99%

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C3G contributes to platelet activation and aggregation by regulating major signaling pathways

Gutiérrez-Herrero

Fernández-Infante

Hernández-Cano

et al. 2020

Sig Transduct Target Ther

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C3G is a GEF (guanine nucleotide exchange factor) for Rap GTPases, among which the isoform Rap1b is an essential protein in platelet biology. Using transgenic mouse models with platelet-specific overexpression of C3G or mutant C3GΔCat, we have unveiled a new function of C3G in regulating the hemostatic function of platelets through its participation in the thrombin-PKC-Rap1b pathway. C3G also plays important roles in angiogenesis, tumor growth, and metastasis through its regulation of the platelet secretome. In addition, C3G contributes to megakaryopoiesis and thrombopoiesis. Here, we used a platelet-specific C3G-KO mouse model to further support the role of C3G in hemostasis. C3G-KO platelets showed a significant delay in platelet activation and aggregation as a consequence of the defective activation of Rap1, which resulted in decreased thrombus formation in vivo. Additionally, we explored the contribution of C3G-Rap1b to platelet signaling pathways triggered by thrombin, PMA or ADP, in the referenced transgenic mouse model, through the use of a battery of specific inhibitors. We found that platelet C3G is phosphorylated at Tyr504 by a mechanism involving PKC-Src. This phosphorylation was shown to be positively regulated by ERKs through their inhibition of the tyrosine phosphatase Shp2. Moreover, C3G participates in the ADP-P2Y12-PI3K-Rap1b pathway and is a mediator of thrombin-TXA 2 activities. However, it inhibits the synthesis of TXA 2 through cPLA 2 regulation. Taken together, our data reveal the critical role of C3G in the main pathways leading to platelet activation and aggregation through the regulation of Rap1b.

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Section: Shp2 Colocalizes With C3g and Inhibits Its Gef Activitysupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

C3G contributes to platelet activation and aggregation by regulating major signaling pathways

Gutiérrez-Herrero

Fernández-Infante

Hernández-Cano

et al. 2020

Sig Transduct Target Ther

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“…Several in vitro studies proposed a role for CalDAG GEFI in ERK1/2 activation [ 10 , 11 , 48 ]. Kawasaki et al found that overexpression of CalDAG GEFI in 293T cells inhibits Elk1 activation and assumed that this would lead to reduced phosphorylation of ERK1/2 further downstream [ 10 ].…”

Section: Discussionmentioning

confidence: 99%

The guanine-nucleotide exchange factor CalDAG GEFI fine-tunes functional properties of regulatory T cells

Niemz

Kliche

Pils

et al. 2017

EuJMI

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Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3– conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI–/– mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI–/– Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI–/– Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI–/– Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.

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“…The rate of Rap1 nucleotide exchange was measured using a fluorescence-based in vitro enzyme assay 17 , 45 , 46 . 100 µL of reaction buffer (20 mM Tris base, 150 mM NaCl, 5 mM MgCl 2 , 2 mM dithiothreitol, 10% [v/v] glycerol, 0.08% [v/v] NP-40, 1 µM Rap1B, 0.1 µM BODIPY-FL-GDP, pH = 7.5) was aliquoted into wells of a Nunc F96 well, black, flat-bottomed plate and the baseline fluorescence intensity (F.I.)…”

Section: Methodsmentioning

confidence: 99%

Two novel, putative mechanisms of action for citalopram-induced platelet inhibition

Roweth

Cook

Moroi

et al. 2018

Sci Rep

Self Cite

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Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram’s ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists.

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A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

Cited by 11 publications

References 29 publications

C3G contributes to platelet activation and aggregation by regulating major signaling pathways

C3G contributes to platelet activation and aggregation by regulating major signaling pathways

The guanine-nucleotide exchange factor CalDAG GEFI fine-tunes functional properties of regulatory T cells

Two novel, putative mechanisms of action for citalopram-induced platelet inhibition

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