RESUMEN
Platelets are small anucleate blood cells supporting vascular function. They circulate in a quiescent state monitoring the vasculature for injuries. Platelets adhere to injury sites and can be rapidly activated to secrete granules and to form platelet/platelet aggregates. These responses are controlled by signalling networks that include G proteins and their regulatory guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Recent proteomics studies have revealed the complete spectrum of G proteins, GEFs, and GAPs present in platelets. Some of these proteins are specific for platelets and very few have been characterised in detail. GEFs and GAPs play a major role in setting local levels of active GTP-bound G proteins in response to activating and inhibitory signals encountered by platelets. Thus, GEFs and GAPs are highly regulated themselves and appear to integrate G protein regulation with other cellular processes. This review focuses on GAPs of small G proteins of the Arf, Rab, Ras, and Rho families, as well as of heterotrimeric G proteins found in platelets.
Asunto(s)
Plaquetas , Proteínas Activadoras de GTPasa , Plaquetas/metabolismo , Humanos , Proteínas Activadoras de GTPasa/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.rpth.2024.102322.].
RESUMEN
Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.
RESUMEN
The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.
Asunto(s)
Células Epiteliales , Glucólisis , Humanos , Glucólisis/genética , Adenosina Trifosfato , Expresión Génica , GlucosaRESUMEN
RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.
Asunto(s)
Proteína Coatómero , Triptófano , Humanos , Proteína Coatómero/química , Proteína Coatómero/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Unión Proteica , Transporte de Proteínas , Proteómica , Triptófano/metabolismoRESUMEN
Phosphorylation is a major regulatory mechanism controlling protein and cell function. Phosphoproteomics is continuing to reveal the extent and complexity of protein phosphorylation. In particular, most proteins are emerging to contain multiple phosphorylation sites. However, phosphoproteomics has outpaced current understanding of the functional roles of individual phospho-sites. In this paper the Phos-tag gel method is presented and discussed in the context of other available tools for phosphorylation research. Strengths and weaknesses of Phos-tag gels are outlined and an integrated approach to phosphorylation research is proposed. SIGNIFICANCE: The Phos-tag gel method has unique strengths especially regarding the analysis of multi-site phosphorylation. A combined approach including Phos-tag gels together with other methods like isotope labelling, phospho-specific antibodies, and mass spectrometry is required to advance current understanding of protein phosphorylation.
Asunto(s)
Fosfoproteínas , Piridinas , Electroforesis en Gel de Poliacrilamida , Geles , Fosfoproteínas/análisis , Fosforilación , Factores de Transcripción/metabolismoRESUMEN
Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.
Asunto(s)
Plaquetas/virología , COVID-19/sangre , Adenosina Trifosfato/metabolismo , Anciano , Coagulación Sanguínea , Plaquetas/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemostasis , Humanos , Inflamación , Unidades de Cuidados Intensivos , Masculino , Volúmen Plaquetario Medio , Persona de Mediana Edad , Selectina-P/sangre , Fenotipo , Factor Plaquetario 4/sangre , Pruebas de Función Plaquetaria , Trombopoyetina/sangreRESUMEN
Breast cancer is the most common female cancer globally, with approximately 12% of patients eventually developing metastatic disease. Critically, limited effective treatment options exist for metastatic breast cancer. Recently, von Willebrand factor (VWF), a haemostatic plasma glycoprotein, has been shown to play an important role in tumour progression and metastasis. In breast cancer, a significant rise in the plasma levels of VWF has been reported in patients with malignant disease compared to benign conditions and healthy controls, with an even greater increase seen in patients with disseminated disease. Direct interactions between VWF, tumour cells, platelets and endothelial cells may promote haematogenous dissemination and thus the formation of metastatic foci. Intriguingly, patients with metastatic disease have unusually large VWF multimers. This observation has been proposed to be a result of a dysfunctional or deficiency of VWF-cleaving protease activity, ADAMTS-13 activity, which may then regulate the platelet-tumour adhesive interactions in the metastatic process. In this review, we provide an overview of VWF in orchestrating the pathological process of breast cancer dissemination, and provide supporting evidence of the role of VWF in mediating metastatic breast cancer.
RESUMEN
BACKGROUND: Circulating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium-derived prostacyclin and nitric oxide stimulate cAMP- and cGMP-dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. OBJECTIVES: We wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide-dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. METHODS: Phosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos-tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull-down assays were performed to analyze protein interaction and function. RESULTS: Our data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA-specific GTPase-activating protein Myo9b and the guanine nucleotide exchange factor GEF-H1. We identify Myo9b S1354 and guanine nucleotide exchange factor-H1 (GEF-H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA-GTP levels. GEF-H1 S886 phosphorylation stimulates binding of 14-3-3ß and has been shown to inhibit GEF function by facilitating binding of GEF-H1 to microtubules. Microtubule disruption increases RhoA-GTP levels confirming the importance of GEF-H1 in platelets. CONCLUSION: Phosphorylation of RhoA regulatory proteins Myo9b and GEF-H1, but not RhoA itself, is involved in cyclic nucleotide-mediated control of RhoA in human platelets.
Asunto(s)
Plaquetas , Miosinas , Nucleótidos Cíclicos , Factores de Intercambio de Guanina Nucleótido Rho , Plaquetas/metabolismo , Células HEK293 , Humanos , Fosforilación , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19-S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)-ENSA/ARPP19-PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.
Asunto(s)
Plaquetas/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Donantes de Sangre , Plaquetas/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células HEK293 , Humanos , Ácido Ocadaico/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/genética , Proteína Fosfatasa 2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , TransfecciónRESUMEN
Platelets are regulated by extracellular cues that impact on intracellular signaling. The endothelium releases prostacyclin and nitric oxide which stimulate the synthesis of cyclic nucleotides cAMP and cGMP leading to platelet inhibition. Other inhibitory mechanisms involve immunoreceptor tyrosine-based inhibition motif-containing receptors, intracellular receptors and receptor desensitization. Inhibitory cyclic nucleotide pathways are traditionally thought to represent a passive background system keeping platelets in a quiescent state. In contrast, cyclic nucleotides are increasingly seen to be dynamically involved in most aspects of platelet regulation. This review focuses on crosstalk between activating and cyclic nucleotide-mediated inhibitory pathways highlighting emerging new hub structures and signaling mechanisms. In particular, interactions of plasma membrane receptors like P2Y12 and GPIb/IX/V with the cyclic nucleotide system are described. Furthermore, differential regulation of the RGS18 complex, second messengers, protein kinases, and phosphatases are presented, and control over small G-proteins by guanine-nucleotide exchange factors and GTPase-activating proteins are outlined. Possible clinical implications of signaling crosstalk are discussed.
RESUMEN
Phos-tag gels are recent tools to dissect protein phosphorylation that operate by inducing a shift in the electrophoretic mobility of phosphorylated proteins compared to their nonphosphorylated counterparts. This article describes the preparation and electrophoresis of Zn2+ -Phos-tag gels along with electrotransfer of the separated phospho- and nonphosphoproteins onto a PVDF membrane using either wet-tank or semidry transfer. We also discuss the theory behind the technology with critical parameters to keep in mind for its successful application. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Fosfoproteínas/análisis , Fosfoproteínas/química , Piridinas/química , Zinc/química , FosforilaciónRESUMEN
Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins.
Asunto(s)
Plaquetas/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Humanos , Activación PlaquetariaRESUMEN
Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.
Asunto(s)
Plaquetas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Proteínas Activadoras de GTPasa/sangre , Células HEK293 , Células HeLa , Humanos , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/sangre , Especificidad por SustratoRESUMEN
Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Proteínas RGS/metabolismo , Proteínas 14-3-3/metabolismo , Línea Celular , Humanos , Inmunoprecipitación , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFß), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis. METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFß induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFß in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFß on mechanically stretched fibroblasts. CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.
RESUMEN
Here we provide evidence that WNT-3a modulates platelet function by regulating the activity of four key GTPase proteins: Rap1, Cdc42, Rac1 and RhoA. We observe WNT-3a to differentially regulate small GTPase activity in platelets, promoting the GDP-bound form of Rap1b to inhibit integrin-α(IIb)ß(3) adhesion, while concomitantly increasing Cdc42 and Rac1-GTP levels thereby disrupting normal platelet spreading. We demonstrate that Daam-1 interacts with Dishevelled upon platelet activation, which correlates with increased RhoA-GTP levels. Upon pre-treatment with WNT-3a, this complex disassociates, concurrent with a reduction in RhoA-GTP. Together these data implicate WNT-3a as a novel upstream regulator of small GTPase activity in platelets.
Asunto(s)
Plaquetas/metabolismo , Regulación de la Expresión Génica , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteína Wnt3A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Plaquetas/citología , Densitometría/métodos , Matriz Extracelular/metabolismo , Guanosina Trifosfato/química , Humanos , Hidrólisis , Proteínas de Microfilamentos , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rhoRESUMEN
Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.
Asunto(s)
Plaquetas/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Activación Plaquetaria/fisiología , Proteínas RGS/metabolismo , Transducción de Señal/fisiología , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosforilación/fisiología , Proteínas RGS/genética , Proteínas RGS/inmunología , Conejos , Receptor Cross-Talk/fisiología , Serina/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Na(+)/I(-) symporter (NIS)-mediated iodide uptake into thyroid follicular cells serves as the basis of radioiodine therapy for thyroid cancer. NIS protein is also expressed in the majority of breast tumors, raising potential for radionuclide therapy of breast cancer. KT5823, a staurosporine-related protein kinase inhibitor, has been shown to increase thyroid-stimulating hormone-induced NIS expression, and thus iodide uptake, in thyroid cells. In this study, we found that KT5823 does not increase but decreases iodide uptake within 0.5 h of treatment in trans-retinoic acid and hydrocortisone-treated MCF-7 breast cancer cells. Moreover, KT5823 accumulates hypoglycosylated NIS, and this effect is much more evident in breast cancer cells than thyroid cells. The hypoglycosylated NIS is core glycosylated, has not been processed through the Golgi apparatus, but is capable of trafficking to the cell surface. KT5823 impedes complex NIS glycosylation at a regulatory point similar to brefeldin A along the N-linked glycosylation pathway, rather than targeting a specific N-glycosylated site of NIS. KT5823-mediated effects on NIS activity and glycosylation are also observed in other breast cancer cells as well as human embryonic kidney cells expressing exogenous NIS. Taken together, KT5823 will serve as a valuable pharmacological reagent to uncover mechanisms underlying differential NIS regulation between thyroid and breast cancer cells at multiple levels.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carbazoles/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Simportadores/metabolismo , Glándula Tiroides/citología , Animales , Brefeldino A/farmacología , Línea Celular , Femenino , Glicosilación , Humanos , Hidrocortisona , Yoduros/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Tirotropina , TretinoinaRESUMEN
Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFkappaB-IkappaBalpha-PKAc complex. We demonstrate mRNA and protein expression for most of the NFkappaB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IkappaBalpha, and conversely, IkappaBalpha was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IkappaBalpha, disruption of an IkappaBalpha-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBalpha phosphorylation, PKA-IkBalpha dissociation, and VASP phosphorylation, and potentiated integrin alphaIIbbeta3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFkappaB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.