Investigating Sema6A‐PlxnA Signaling Mechanisms in Development: Identification of Key PlxnA Phosphorylation Sites and the Discovery of a Novel Naturally‐Released Sema
Authors: St. Clair Riley , Emerson Sarah , D'Elia Kristen , Weir Marion , Schmoker Anna , Williams Kori , Goldstein Matthew , Stant Elizabeth , Ebert Alicia , Ballif Bryan 
Published: June 2021

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in the developing nervous system, cardiovasculature, and immune system. Semas signal predominantly through Plexin (Plxn) receptors to regulate cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. We have previously shown that the transmembrane Sema6A and its PlxnA2 receptor are critical in vertebrate eye development, as morpholino‐based knock‐down of these proteins in zebrafish results in decreased early eye field cohesion, smaller eye size, and impaired retinal lamination. Due to the transmembrane nature of Sema6A, reverse signaling can occur and it is thought to contribute to the observed phenotypes. Although several cellular players governing Sema‐Plxn signaling have been identified, the molecular mechanisms that initiate forward and reverse signaling are only partially understood. Therefore, we aimed to investigate the receptor‐proximal events of Sema‐Plxn signaling and we report here two main findings towards a better understanding of the early events of Sema‐Plxn signaling: 1) PlxnA receptor phosphorylation at two tyrosine sites is critical for zebrafish eye development and 2) Sema6A has a functional naturally‐released ectodomain, sSema6A.We used mass spectrometry‐based approaches to identify highly‐conserved, Fyn kinase‐mediated PlxnA tyrosine phosphorylation sites. Mutation of these sites to phenylalanine results in significantly decreased Fyn‐dependent PlxnA tyrosine phosphorylation. Furthermore, in contrast to wildtype human PLXNA2 mRNA, mRNA harboring these point mutations cannot rescue eye developmental defects when co‐injected with a plxnA2 morpholino in zebrafish embryos. Interestingly, while investigating whether or not these sites are phosphorylated upon Sema6A ligand binding, we serendipitously discovered that a functional soluble Sema6A, sSema6A, is spontaneously released from cells expressing the full‐length transmembrane Sema6A. Using zebrafish eye explants, we show that sSema6A promotes early eye field cohesion, a process known to be Sema6A‐dependent. While other soluble Sema ectodomains have been identified in the immune system, we describe here the first soluble ectodomain from a transmembrane Sema that has predominant roles in the nervous system. Together these data suggest that Sema6A may have long‐range effects in addition to its canonical contact‐mediated functions and that Fyn‐dependent phosphorylation is a key feature of vertebrate PlxnA signal transduction. Future work will investigate if the transmembrane and secreted forms of Sema6A can induce Fyn‐mediated PlxnA tyrosine phosphorylation.Support or Funding InformationNational Science Foundation (NSF) IOS grants 1021795 and 1625154; NSF DBI REU grant 1262786;the Vermont Genetics Network through U. S. National Institutes of Health (NIH) Grant 8P20GM103449 from the INBRE program of the NIGMS; and U.S. NIH Grant 5P20RR016435 fromthe COBRE program of the NIGMS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.