Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets
2022; Elsevier BV; Volume: 298; Issue: 8 Linguagem: Inglês
10.1016/j.jbc.2022.102189
ISSN1083-351X
AutoresJohn C. Kostyak, Benjamin Mauri, Carol Dangelmaier, Hymavathi Reddy Vari, Akruti Patel, Monica Wright, Haritha Reddy, Alexander Y. Tsygankov, Satya P. Kunapuli,
Tópico(s)Autoimmune Bullous Skin Diseases
ResumoImmune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal. Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal. Immune cells express several receptors that, upon activation, their cytoplasmic tails are phosphorylated on tyrosines in a stretch of domain known as immune tyrosine activation motif (ITAM) containing two YXX(L/I) motifs (1Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. et al.The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.J. Biol. 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There also exists a different group of receptors that contain only one YXX(L/I) motif in the cytoplasmic domain that is known as hemITAM (1Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. et al.The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 2Underhill D.M. Goodridge H.S. The many faces of ITAMs.Trends Immunol. 2007; 28: 66-73Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The hemITAM-containing receptors include the C-type lectin-like receptor-2 (CLEC-2) on platelets and immune cells as well as dectin in macrophages (1Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. et al.The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.J. Biol. 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Navarro-Nunez L. Nash C.A. Mourao-Sa D. Eble J.A. et al.Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling.J. Biol. Chem. 2011; 286: 4107-4116Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 4Watson S.P. Asazuma N. Atkinson B. Berlanga O. Best D. Bobe R. et al.The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen.Thromb. Haemost. 2001; 86: 276-288Crossref PubMed Scopus (118) Google Scholar, 5Watson S.P. Auger J.M. McCarty O.J. Pearce A.C. GPVI and integrin alphaIIb beta3 signaling in platelets.J. Thromb. Haemost. 2005; 3: 1752-1762Crossref PubMed Scopus (326) Google Scholar), the mechanisms subsequent to its binding leading to its activation are not clear. Syk contains several tyrosine residues that are known to be phosphorylated upon its binding to the phosphorylated ITAM or hemITAM (6Geahlen R.L. Syk and pTyr'd: signaling through the B cell antigen receptor.Biochim. 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Considering that platelets express both ITAM-containing and hemITAM-containing receptors and Syk plays a key role in platelet signaling and activation (9Bauer M. Maschberger P. Quek L. Briddon S.J. Dash D. Weiss M. et al.Genetic and pharmacological analyses of involvement of Src-family, Syk and Btk tyrosine kinases in platelet shape change. Src-kinases mediate integrin alphaIIb beta3 inside-out signalling during shape change.Thromb. Haemost. 2001; 85: 331-340Crossref PubMed Scopus (29) Google Scholar, 10Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (257) Google Scholar, 11Manne B.K. Getz T.M. Hughes C.E. Alshehri O. Dangelmaier C. Naik U.P. et al.Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2).J. Biol. 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Those that signal through a nonreceptor tyrosine kinase include the ITAM-bearing GPVI receptor and the Fc receptor for immunoglobulin, FcγRIIA, as well as the hemITAM-bearing receptor CLEC-2. Ligand engagement of these receptors results in Src family kinase (SFK) phosphorylation of tyrosine residues within the cytoplasmic domain of the receptor (10Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (257) Google Scholar, 17Watson S.P. Gibbins J. Collagen receptor signalling in platelets: extending the role of the ITAM.Immunol. Today. 1998; 19: 260-264Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Quek L.S. Pasquet J.M. Hers I. Cornall R. Knight G. 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Hartwig J.H. et al.CLEC-2 activates Syk through dimerization.Blood. 2010; 115: 2947-2955Crossref PubMed Scopus (117) Google Scholar). This elicits a downstream signaling cascade that results in platelet functional responses. While many similarities between GPVI and CLEC-2 are present, signaling events leading to Syk phosphorylation are not identical between the two receptors (29Manne B.K. Badolia R. Dangelmaier C. Eble J.A. Ellmeier W. Kahn M. et al.Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets.J. Biol. Chem. 2015; 290: 11557-11568Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, in either case, Syk is required.Syk is phosphorylated on several tyrosine residues in response to GPVI or CLEC-2 engagement. We previously showed that several tyrosine residues on Syk are phosphorylated in response to the GPVI agonist convulxin (8Reppschlager K. Gosselin J. Dangelmaier C.A. Thomas D.H. Carpino N. McKenzie S.E. et al.TULA-2 protein phosphatase suppresses activation of Syk through the GPVI platelet receptor for collagen by dephosphorylating Tyr(P)346, a regulatory site of Syk.J. Biol. Chem. 2016; 291: 22427-22441Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 31Chen X. Ren L. Kim S. Carpino N. Daniel J.L. Kunapuli S.P. et al.Determination of the substrate specificity of protein-tyrosine phosphatase TULA-2 and identification of Syk as a TULA-2 substrate.J. Biol. Chem. 2010; 285: 31268-31276Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Furthermore, we and others have demonstrated that both Syk Y519/520 and Y346 are phosphorylated downstream of CLEC-2 (32Kostyak J.C. Liverani E. Kunapuli S.P. PKC-epsilon deficiency alters progenitor cell populations in favor of megakaryopoiesis.PLoS One. 2017; 12e0182867Crossref PubMed Scopus (3) Google Scholar, 33Hughes C.E. Finney B.A. Koentgen F. Lowe K.L. Watson S.P. 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The molecular basis of their functions is not fully understood, but it appears likely that phosphorylation of these residues is involved in the transition of Syk from its autoinhibited to its active conformation (44Gradler U. Schwarz D. Dresing V. Musil D. Bomke J. Frech M. et al.Structural and biophysical characterization of the Syk activation switch.J. Mol. Biol. 2013; 425: 309-333Crossref PubMed Scopus (54) Google Scholar). Although some data argue that Y342 is the most important of the regulatory tyrosine residues in the interdomain B (40Zhang J. Berenstein E. Siraganian R.P. Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells.Mol. Cell. Biol. 2002; 22: 8144-8154Crossref PubMed Scopus (50) Google Scholar, 41Simon M. Vanes L. Geahlen R.L. Tybulewicz V.L. Distinct roles for the linker region tyrosines of Syk in FcepsilonRI signaling in primary mast cells.J. Biol. 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In this report, we demonstrate that phosphorylation of Y342 on Syk plays an important positive regulatory role in the signal transduction from an ITAM or a hemITAM receptor. We will also show that bleeding is unaffected in Syk Y342F mice while thrombus formation is significantly prolonged.ResultsProduction and characterization of Syk Y342F knock-in micePrimary mast cells and basophilic cells in culture expressing Syk Y342F as a result of transfection or transduction demonstrated that tyrosine phosphorylation of mutated Syk and proteins important for ITAM-mediated signaling was reduced as compared with cells expressing WT Syk (40Zhang J. Berenstein E. Siraganian R.P. Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells.Mol. Cell. Biol. 2002; 22: 8144-8154Crossref PubMed Scopus (50) Google Scholar, 41Simon M. Vanes L. Geahlen R.L. Tybulewicz V.L. Distinct roles for the linker region tyrosines of Syk in FcepsilonRI signaling in primary mast cells.J. Biol. Chem. 2005; 280: 4510-4517Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, to determine the function of Y342 in downstream signaling of hemITAM receptors and ITAM receptors, and phosphorylation of other sites on Syk, we produced an Syk Y342F knock-in mouse using the CRSPR/Cas9 technique through a commercial source (Cyagen). The Syk Y342F mutation was confirmed by both sequencing and digesting the PCR product with a restriction enzyme, RsaI. The oligonucleotides used for PCR are forward primer 5′-CTCCGCTGCATGCAACTGTC and reverse primer 5′-GCAGTGCAATGAGTCAACGGTGC. RsaI can cleave the WT nucleotide sequence (gtac) but cannot cleave the knock-in sequence (gttc). Therefore, the results after the digest are as follows: WT (+/+) 166 & 155 bp products, heterozygous (+/−) 155, 166, and 317 bp products, and homozygous (−/−) 317 bp product. An example of these PCR products is shown in Figure 1A. We also confirmed that the genotyping using sequencing of the PCR product (Fig. 1B). To confirm the Y342F mutation at the protein level, we isolated platelets from Syk Y342F and WT littermate control mice and stimulated them with collagen-related peptide (CRP). We performed Western blot analysis to visualize phosphorylation of Y342. We observed a robust band in WT mouse platelets treated with CRP at any concentration, but no band is present at the expected molecular weight in samples obtained from Syk Y342F mouse platelets (Fig. 1C). These data demonstrate that we successfully produced Syk Y342F knock-in mice.Syk Y342F knock-in mice are viableHeterozygous Syk Y342F knock-in mice and homozygous Syk Y342F knock-in mice bred normally and produced pups at expected Mendelian ratios. In contrast, Syk knockout mice are not viable and die perinatally (45Cheng A.M. Rowley B. Pao W. Hayday A. Bolen J.B. Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development.Nature. 1995; 378: 303-306Crossref PubMed Scopus (546) Google Scholar, 46Turner M. Mee P.J. Costello P.S. Williams O. Price A.A. Duddy L.P. et al.Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.Nature. 1995; 378: 298-302Crossref PubMed Scopus (643) Google Scholar). Blood cell counts were not altered in Syk Y342F knock-in mice except for significantly reduced circulating red blood cells (Table 1). As all other cell counts were normal, we chose not to pursue the red blood cell number reduction during this study. However, it is an interesting finding and would be worth investigating later. Platelets from Syk Y342F mice respond normally to the G protein–coupled receptor agonists AYPGKF (PAR4) and 2-MeSADP (P2Y) (Fig. 2).Table 1Blood cell counts from Syk Y342F and WT littermate control miceGenotypeWBC (K/ml)NE (K/ml)LY (K/ml)RBC (M/ml)PLT (K/ml)MPV (fl)SykWT/WT8.86 ± 0.800.92 ± 0.127.44 ± 0.719.76 ± 0.23692.48 ± 20.794.07 ± 0.05SykY342F/Y342F10.54 ± 1.090.63 ± 0.139.22 ± 0.858.40 ± 0.13ap < 0.05 compared with WT, n = 12.710.44 ± 66.023.98 ± 0.05Abbreviations: LY, lymphocyte; MPV, mean platelet volume; NE, neutrophil; PLT, platelet; RBC, red blood cell; WBC, white blood cell.a p < 0.05 compared with WT, n = 12. Open table in a new tab Figure 2GPCR-mediated platelet reactivity is intact in Syk Y342F mice. A, representative aggregation and secretion tracings of platelets from WT and Syk Y342F mice stimulated with the PAR4 agonist AYPGKF. B, quantitation of aggregation and C, secretion of platelets from Syk Y342F and WT littermate control mice stimulated with 100 μM AYPGKF. D, representative aggregation and secretion tracings of platelets from WT and Syk Y342F mice stimulated with the P2Y receptor agonist 2-MeSADP. E, quantitation of aggregation and F, secretion of platelets from Syk Y342F and WT mice stimulate
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