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Thromb Haemost 2024; 124(10): 937-947
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).
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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

 

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