The RhoA regulators Myo9b and GEF-H1 are targets of cyclic nucleotide-dependent kinases in platelets.
14-3-3 proteins
GTPase-activating proteins
cyclic AMP-dependent protein kinases
cyclic GMP-dependent protein kinases
guanine nucleotide exchange factors
phosphorylation
Journal
Journal of thrombosis and haemostasis : JTH
ISSN: 1538-7836
Titre abrégé: J Thromb Haemost
Pays: England
ID NLM: 101170508
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
30
03
2020
revised:
15
06
2020
accepted:
13
07
2020
pubmed:
22
7
2020
medline:
15
5
2021
entrez:
22
7
2020
Statut:
ppublish
Résumé
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. We wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide-dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. 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. 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 14-3-3β 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. 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.
Sections du résumé
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 14-3-3β 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.
Identifiants
pubmed: 32692911
doi: 10.1111/jth.15028
pii: S1538-7836(22)03718-7
doi:
Substances chimiques
ARHGEF2 protein, human
0
Nucleotides, Cyclic
0
Rho Guanine Nucleotide Exchange Factors
0
myosin IXB
0
RHOA protein, human
124671-05-2
Myosins
EC 3.6.4.1
rhoA GTP-Binding Protein
EC 3.6.5.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3002-3012Informations de copyright
© 2020 International Society on Thrombosis and Haemostasis.
Références
NagyZ, SmolenskiA. Cyclic nucleotide-dependent inhibitory signaling interweaves with activating pathways to determine platelet responses. Res Pract Thromb Haemost. 2018;2(3):558-571.
RaslanZ, NaseemKM. The control of blood platelets by cAMP signalling. Biochem Soc Trans. 2014;42(2):289-294.
MakhoulS, WalterE, PagelO, et al. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets. Nitric Oxide. 2018;76:71-80.
BeckF, GeigerJ, GambaryanS, et al. Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways. Blood. 2014;123(5):e1-e10.
KlagesB, BrandtU, SimonMI, SchultzG, OffermannsS. Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol. 1999;144(4):745-754.
PleinesI, HagedornI, GuptaS, et al. Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood. 2012;119(4):1054-1063.
AslanJE, McCartyOJ. Rho GTPases in platelet function. J Thromb Haemost. 2013;11(1):35-46.
GratacapM-P, PayrastreB, NieswandtB, OffermannsS. Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides. J Biol Chem. 2001;276(51):47906-47913.
SuzukiA, ShinJ-W, WangY, et al. RhoA is essential for maintaining normal megakaryocyte ploidy and platelet generation. PLoS One. 2013;8(7):1-9.
EllerbroekSM, WennerbergK, BurridgeK. serine phosphorylation negatively regulates RhoA in vivo. J Biol Chem. 2003;278(21):19023-19031.
AburimaA, WraithKS, RaslanZ, LawR, MagwenziS, NaseemKM. cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway. Blood. 2013;122(20):3533-3545.
AburimaA, WalladbegiK, WakeJD, NaseemKM. cGMP signaling inhibits platelet shape change through regulation of the RhoA-Rho Kinase-MLC phosphatase signaling pathway. J Thromb Haemost. 2017;15(8):1668-1678.
AtkinsonL, YusufMZ, AburimaA, et al. Reversal of stress fibre formation by nitric oxide mediated RhoA inhibition leads to reduction in the height of preformed thrombi. Sci Rep. 2018;8. https://doi.org/10.1038/s41598-018-21167-6
BeckF, GeigerJ, GambaryanS, et al. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Blood. 2017;129(2):e1-e12.
NagyZ, WynneK, vonKriegsheimA, GambaryanS, SmolenskiA. Cyclic nucleotide-dependent protein kinases target ARHGAP17 and ARHGEF6 complexes in platelets. J Biol Chem. 2015;290(50):29974-29983.
GegenbauerK, EliaG, Blanco-FernandezA, SmolenskiA. Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets. Blood. 2012;119(16):3799-3807.
HoffmeisterM, RihaP, NeumüllerO, DanielewskiO, SchultessJ, SmolenskiAP. Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets. J Biol Chem. 2008;283(4):2297-2306.
ElversM. RhoGAPs and Rho-GTPases in platelets. Hamostaseologie. 2015;36(03):168-177.
GoggsR, WilliamsCM, MellorH, PooleAW. Platelet Rho GTPases-a focus on novel players, roles and relationships. Biochem J. 2015;466(3):431-442.
MüllerRT, HonnertU, ReinhardJ, BählerM. The rat myosin myr 5 is a GTPase-activating protein for Rho in vivo: essential role of arginine 1695. Mol Biol Cell. 1997;8(10):2039-2053.
KrendelM, ZenkeFT, BokochGM. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat Cell Biol. 2002;4:294-301.
vonThunA, PreisingerC, RathO, et al. Extracellular signal-regulated kinase regulates RhoA activation and tumor cell plasticity by inhibiting guanine exchange factor H1 activity. Mol Cell Biol. 2013;33(22):4526-4537.
van denBoomF, DüssmannH, UhlenbrockK, AbouhamedM, BählerM. The myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization. Mol Biol Cell. 2007;18(4):1507-1518.
NagyZ, ComerS, SmolenskiA. Analysis of protein phosphorylation using Phos-tag gels. Curr Protocols Protein Sci. 2018;93(1):1-10.
SchneiderCA, RasbandWS, EliceiriKW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-675.
QiaoJ, HuangF, LumH. PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. 2003;284(6):L972-L980.
AltschulerD, LapetinaEG. Mutational analysis of the cAMP-dependent protein kinase-mediated phosphorylation site of Rap1b. J Biol Chem. 1993;268(10):7527-7531.
TakahashiM, LiY, DillonTJ, StorkPJS. Phosphorylation of Rap1 by cAMP-dependent protein kinase (PKA) creates a binding site for KSR to sustain ERK activation by cAMP. J Biol Chem. 2017;292(4):1449-1461.
KennellyPJ, KrebsEG. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991;266(24):15555-15558.
HornbeckPV, ZhangB, MurrayB, KornhauserJM, LathamV. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015;43(D1):D512-D520.
SieversF, WilmA, DineenD, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539.
MeiriD, GreeveMA, BrunetA, et al. Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol Cell Biol. 2009;29(21):5963-5973.
ChangY-C, NalbantP, BirkenfeldJ, ChangZ-F, BokochGM. GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA. Mol Biol Cell. 2008;19(5):2147-2153.
ZenkeFT, KrendelM, DerMardirossianC, KingCC, BohlBP, BokochGM. p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized rho exchange factor. J Biol Chem. 2004;279(18):18392-18400.
AzoiteiML, NohJ, MarstonDJ, et al. Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways. J Cell Biol. 2019;218(9):3077-3097.
SauzeauV, Le JeuneH, Cario-ToumaniantzC, et al. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem. 2000;275(28):21722-21729.
SawadaN, ItohH, YamashitaJ, et al. cGMP-dependent protein kinase phosphorylates and inactivates RhoA. Biochem Biophys Res Comm. 2001;280(3):798-805.
SmolenskiA. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost. 2012;10(2):167-176.
BolzS-S, VogelL, SollingerD, et al. Nitric oxide-induced decrease in calcium sensitivity of resistance arteries is attributable to activation of the myosin light chain phosphatase and antagonized by the RhoA/Rho kinase pathway. Circulation. 2003;107(24):3081-3087.
OishiA, MakitaN, SatoJ, IiriT. Regulation of RhoA signaling by the cAMP-dependent phosphorylation of RhoGDIα. J Biol Chem. 2012;287(46):38705-38715.
BagciH, SriskandarajahN, RobertA, et al. Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms. Nat Cell Biol. 2020;22(1):120-134.
LiaoW, ElfrinkK, BählerM. Head of Myosin IX binds calmodulin and moves processively toward the Plus-end of actin filaments. J Biol Chem. 2010;285(32):24933-24942.
HanleyPJ, XuY, KronlageM, et al. Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility. Proc Natl Acad Sci USA. 2010;107(27):12145-12150.
MoalliF, FichtX, GermannP, et al. The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8(+) T cells. J Exp Med. 2018;215(7):1869-1890.
ChandhokeSK, MoosekerMS. A role for myosin IXb, a motor-RhoGAP chimera, in epithelial wound healing and tight junction regulation. Mol Biol Cell. 2012;23(13):2468-2480.
MészárosB, ErdosG, DosztányiZ. IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding. Nucleic Acids Res. 2018;46(W1):W329-W337.
BahA, Forman-KayJD. Modulation of intrinsically disordered protein function by post-translational modifications. J Biol Chem. 2016;291(13):6696-6705.
GegenbauerK, NagyZ, SmolenskiA. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets. PLoS One. 2013;8(11):e80251.1-10.
LévayM, SettlemanJ, LigetiE. Regulation of the substrate preference of p190RhoGAP by protein kinase C-mediated phosphorylation of a phospholipid binding site. Biochemistry. 2009;48(36):8615-8623.
GambaryanS, KobsarA, RukoyatkinaN, et al. Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of protein kinase A from an NFkappaB-IkappaB complex. J Biol Chem. 2010;285(24):18352-18363.
AslanJE, BakerSM, LorenCP, et al. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation. Am J Physiol Cell Physiol. 2013;305(5):C519-C528.
GaoY, SmithE, KerE, et al. Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes. Dev Cell. 2012;22(3):573-584.
YamahashiY, SaitoY, Murata-KamiyaN, HatakeyamaM. Polarity-regulating kinase partitioning-defective 1b (PAR1b) phosphorylates guanine nucleotide exchange factor H1 (GEF-H1) to regulate RhoA-dependent actin cytoskeletal reorganization. J Biol Chem. 2011;286(52):44576-44584.
BirkenfeldJ, NalbantP, BohlBP, PertzO, HahnKM, BokochGM. GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases. Dev Cell. 2007;12(5):699-712.
MeiriD, MarshallCB, MokadyD, et al. Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1. Nat Commun. 2014;5:1-14.
GraesslM, KochJ, CalderonA, et al. An excitable Rho GTPase signaling network generates dynamic subcellular contraction patterns. J Cell Biol. 2017;216(12):4271-4285.