Download PDF
Thromb Haemost
DOI: 10.1055/a-2332-6321
Cellular Haemostasis and Platelets

The LAT Rheostat as a Regulator of Megakaryocyte Activation

Alyssa J. Moroi 
1   Blood Research Institute, Versiti Blood Center of Wisconsin, Milwaukee, Wisconsin, United States
,
Peter J. Newman
1   Blood Research Institute, Versiti Blood Center of Wisconsin, Milwaukee, Wisconsin, United States
2   Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3   Department of Cell Biology, Neurobiology and, Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
› Author Affiliations Funding This work was supported by NIH grant R35 HL-139937 (to P.J.N).
› Further Information
Also available at
    Permissions and Reprints


Abstract

Background Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132—the binding site for PLC-γ2—is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known.

Methods To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences.

Results iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced.

Conclusion These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

Keywords

GPVI - induced pluripotent stem cell - megakaryocytes - platelets - signal transduction

Authors' Contribution

A.J.M. designed and performed experiments, analyzed data, made the figures, and wrote the manuscript. P.J.N. designed experiments, wrote the manuscript, and supervised the work.


Supplementary Material



Publication History

Received: 29 November 2023

Accepted: 23 May 2024

Accepted Manuscript online:
24 May 2024

Article published online:
14 June 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Stuttgart · New York

 

  • References

  • 1 Rayes J, Watson SP, Nieswandt B. Functional significance of the platelet immune receptors GPVI and CLEC-2. J Clin Invest 2019; 129 (01) 12-23
    CrossrefPubMedGoogle Scholar
  • 2 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
    CrossrefPubMedGoogle Scholar
  • 3 Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett 1997; 413 (02) 255-259
    CrossrefPubMedGoogle Scholar
  • 4 Tsuji M, Ezumi Y, Arai M, Takayama H. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J Biol Chem 1997; 272 (38) 23528-23531
    CrossrefPubMedGoogle Scholar
  • 5 Quek LS, Pasquet JM, Hers I. et al. Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway. Blood 2000; 96 (13) 4246-4253
    CrossrefPubMedGoogle Scholar
  • 6 Jiang Y, Cheng H. Evidence of LAT as a dual substrate for Lck and Syk in T lymphocytes. Leuk Res 2007; 31 (04) 541-545
    CrossrefPubMedGoogle Scholar
  • 7 Paz PE, Wang S, Clarke H, Lu X, Stokoe D, Abo A. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells. Biochem J 2001; 356 (Pt 2): 461-471
    CrossrefPubMedGoogle Scholar
  • 8 Pasquet JM, Gross B, Quek L. et al. LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI. Mol Cell Biol 1999; 19 (12) 8326-8334
    CrossrefPubMedGoogle Scholar
  • 9 Wonerow P, Watson SP. The transmembrane adapter LAT plays a central role in immune receptor signalling. Oncogene 2001; 20 (44) 6273-6283
    CrossrefPubMedGoogle Scholar
  • 10 Asazuma N, Wilde JI, Berlanga O. et al. Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin. J Biol Chem 2000; 275 (43) 33427-33434
    CrossrefPubMedGoogle Scholar
  • 11 Yablonski D. Bridging the gap: modulatory roles of the Grb2-Family adaptor, gads, in cellular and allergic immune responses. Front Immunol 2019; 10: 1704
    CrossrefPubMedGoogle Scholar
  • 12 Kalia N, Auger JM, Atkinson B, Watson SP. Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo. Microcirculation 2008; 15 (04) 325-335
    CrossrefPubMedGoogle Scholar
  • 13 Hughes CE, Auger JM, McGlade J, Eble JA, Pearce AC, Watson SP. Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC-2. J Thromb Haemost 2008; 6 (12) 2152-2159
    CrossrefPubMedGoogle Scholar
  • 14 Ragab A, Séverin S, Gratacap MP. et al. Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation: insights into the mechanism of PLC gamma 2 activation. Blood 2007; 110 (07) 2466-2474
    CrossrefPubMedGoogle Scholar
  • 15 Wang H, Kadlecek TA, Au-Yeung BB. et al. ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol 2010; 2 (05) a002279
    CrossrefPubMedGoogle Scholar
  • 16 Zhang W, Trible RP, Zhu M, Liu SK, McGlade CJ, Samelson LE. Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J Biol Chem 2000; 275 (30) 23355-23361
    CrossrefPubMedGoogle Scholar
  • 17 Zhu M, Janssen E, Zhang W. Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J Immunol 2003; 170 (01) 325-333
    CrossrefPubMedGoogle Scholar
  • 18 Shah NH, Wang Q, Yan Q. et al. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor. eLife 2016; 5: e20105
    CrossrefPubMedGoogle Scholar
  • 19 Courtney AH, Lo WL, Weiss A. TCR signaling: mechanisms of initiation and propagation. Trends Biochem Sci 2018; 43 (02) 108-123
    CrossrefPubMedGoogle Scholar
  • 20 Lin J, Weiss A. Identification of the minimal tyrosine residues required for linker for activation of T cell function. J Biol Chem 2001; 276 (31) 29588-29595
    CrossrefPubMedGoogle Scholar
  • 21 Zheng Y, Adams T, Zhi H. et al. Restoration of responsiveness of phospholipase Cγ2-deficient platelets by enforced expression of phospholipase Cγ1. PLoS One 2015; 10 (03) e0119739
    CrossrefPubMedGoogle Scholar
  • 22 Lo WL, Shah NH, Rubin SA. et al. Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination. Nat Immunol 2019; 20 (11) 1481-1493
    CrossrefPubMedGoogle Scholar
  • 23 Arbulo-Echevarria MM, Vico-Barranco I, Narbona-Sánchez I, García-Cózar F, Miazek A, Aguado E. Increased protein stability and interleukin-2 production of a LATG131D variant with possible implications for T cell anergy. Front Cell Dev Biol 2020; 8: 561503
    CrossrefPubMedGoogle Scholar
  • 24 Moroi AJ, Zwifelhofer NM, Riese MJ, Newman DK, Newman PJ. Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation. Blood Adv 2019; 3 (07) 1154-1166
    CrossrefPubMedGoogle Scholar
  • 25 Moroi AJ, Newman PJ. Conditional CRISPR-mediated deletion of Lyn kinase enhances differentiation and function of iPSC-derived megakaryocytes. J Thromb Haemost 2022; 20 (01) 182-195
    CrossrefPubMedGoogle Scholar
  • 26 Ito Y, Nakamura S, Sugimoto N. et al. Turbulence activates platelet biogenesis to enable clinical scale ex vivo production. Cell 2018; 174 (03) 636-648.e18
    CrossrefPubMedGoogle Scholar
  • 27 Thon JN, Mazutis L, Wu S. et al. Platelet bioreactor-on-a-chip. Blood 2014; 124 (12) 1857-1867
    CrossrefPubMedGoogle Scholar
  • 28 Montenont E, Bhatlekar S, Jacob S. et al. CRISPR-edited megakaryocytes for rapid screening of platelet gene functions. Blood Adv 2021; 5 (09) 2362-2374
    CrossrefPubMedGoogle Scholar
  • 29 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8 (11) 2281-2308
    CrossrefPubMedGoogle Scholar
  • 30 Zhang N, Newman PJ. Packaging functionally important plasma proteins into the α-granules of human-induced pluripotent stem cell-derived megakaryocytes. J Tissue Eng Regen Med 2019; 13 (02) 244-252
    CrossrefPubMedGoogle Scholar
  • 31 Yablonski D, Kuhne MR, Kadlecek T, Weiss A. Uncoupling of nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell. Science 1998; 281 (5375) 413-416
    CrossrefPubMedGoogle Scholar
  • 32 Braiman A, Barda-Saad M, Sommers CL, Samelson LE. Recruitment and activation of PLCgamma1 in T cells: a new insight into old domains. EMBO J 2006; 25 (04) 774-784
    CrossrefPubMedGoogle Scholar
  • 33 Balagopalan L, Kortum RL, Coussens NP, Barr VA, Samelson LE. The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters. J Biol Chem 2015; 290 (44) 26422-26429
    CrossrefPubMedGoogle Scholar
  • 34 Horejsí V, Zhang W, Schraven B. Transmembrane adaptor proteins: organizers of immunoreceptor signalling. Nat Rev Immunol 2004; 4 (08) 603-616
    CrossrefPubMedGoogle Scholar
  • 35 Grädler U, Schwarz D, Dresing V. et al. Structural and biophysical characterization of the Syk activation switch. J Mol Biol 2013; 425 (02) 309-333
    CrossrefPubMedGoogle Scholar
  • 36 Yan Q, Barros T, Visperas PR. et al. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol Cell Biol 2013; 33 (11) 2188-2201
    CrossrefPubMedGoogle Scholar
  • 37 Xue L, Wang WH, Iliuk A. et al. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates. Proc Natl Acad Sci U S A 2012; 109 (15) 5615-5620
    CrossrefPubMedGoogle Scholar
  • 38 Chu DH, Spits H, Peyron JF, Rowley RB, Bolen JB, Weiss A. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling. EMBO J 1996; 15 (22) 6251-6261
    CrossrefPubMedGoogle Scholar
  • 39 Williams BL, Schreiber KL, Zhang W. et al. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line. Mol Cell Biol 1998; 18 (03) 1388-1399
    CrossrefPubMedGoogle Scholar
  • 40 Houtman JC, Barda-Saad M, Samelson LE. Examining multiprotein signaling complexes from all angles. FEBS J 2005; 272 (21) 5426-5435
    CrossrefPubMedGoogle Scholar
  • 41 Perez-Villar JJ, Whitney GS, Sitnick MT. et al. Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. Biochemistry 2002; 41 (34) 10732-10740
    CrossrefPubMedGoogle Scholar
  • 42 Miles J, Bailey SL, Obenaus AM. et al. Storage temperature determines platelet GPVI levels and function in mice and humans. Blood Adv 2021; 5 (19) 3839-3849
    CrossrefPubMedGoogle Scholar
  • 43 Hosseini E, Ghasemzadeh M, Nassaji F, Jamaat ZP. GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion. Platelets 2017; 28 (05) 498-508
    CrossrefPubMedGoogle Scholar