The LAT rheostat as a regulator of megakaryocyte activation

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 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 iPS cells, differentiated them into megakaryocytes, and examined its functional consequences. Results: iPS cells expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to 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.