The SH3 domain of Fyn kinase interacts with and induces liquid–liquid phase separation of the low-complexity domain of hnRNPA2

Joshua Amaya; Veronica H. Ryan; Nicolas L. Fawzi

doi:10.1074/jbc.RA118.005120

The SH3 domain of Fyn kinase interacts with and induces liquid–liquid phase separation of the low-complexity domain of hnRNPA2

From the ^‡Department of Molecular Pharmacology, Physiology, and Biotechnology and
the ^§Graduate Program in Neuroscience, Brown University, Providence, Rhode Island 02912

↵4 To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912. Tel.: 401-863-5232; Fax: 401-863-6087; E-mail: nicolas_fawzi{at}brown.edu.

↵1 Both authors contributed equally to this work.
Edited by Norma M. Allewell

Abstract

Liquid–liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3-binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein.

Footnotes

↵2 Supported in part by two summer Karen T. Romer Undergraduate Teaching and Research Awards.
↵3 Supported in part by T32MH020068, a Graduate Award from the Robert J. and Nancy D. Carney Institute for Brain Science at Brown University, and NINDS, National Institutes of Health, Grant F31NS110301.
This work was supported in part by NIGMS, National Institutes of Health, Grants R01GM118530 (to N. L. F.) and P20GM104937. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
This article was selected as one of our Editors' Picks.
This article contains Figs. S1–S3.
The NMR spectroscopy data reported in this paper have been submitted to the Biological Magnetic Resonance Data Bank (BMRB) under BMRB accession number 27649.