RT Journal Article
SR Electronic
T1 Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 598201
DO 10.1101/598201
A1 Marc D. Panas
A1 Nancy Kedersha
A1 Tim Schulte
A1 Rui M. Branca
A1 Pavel Ivanov
A1 Paul Anderson
YR 2019
UL http://biorxiv.org/content/early/2019/04/09/598201.abstract
AB Stress granules (SGs) are cytoplasmic, non-membranous RNA/protein structures that assemble in response to environmental stress. G3BP is a critical SG-nucleating protein, and its ability to regulate SGs has been reported to be regulated by serine 149 phosphorylation. We now report that the constructs engineered to contain non-phosphorylatable and phosphomimetic (G3BP1-S149A and G3BP1-S149E, respectively) mutations used in many studies include additional unintended mutations (A54T/S149A and S99P/S149E) one of which (S99P) is responsible for the effects on SG assembly attributed to S149E. Specifically, the S99P mutation alone reduces SG nucleation and impairs the ability to rescue SG assembly in ΔΔG3BP1/2 U2OS KO cells, challenging the widely-stated conclusion that de-phosphorylation of serine 149 in G3BP1 promotes SG assembly. We used comparative mass spectrometry analysis of both (1) ectopically expressed GFP-G3BP1 in ΔΔG3BP1/2 U2OS KO and (2) endogenous G3BP1 in wild-type U2OS, with and without sodium arsenite treatment, in an attempt to reproduce earlier findings, but found no significant changes in S149 phosphorylation that correlate with arsenite-induced SG formation.MSmass spectrometrySAsodium arseniteCZclotrimazoleeIFeukaryotic initiation factorIPimmunoprecipitateKOknockoutPat APateamine Ap-eIF2αphospho-eIF2αSGstress granulewtwild typeNTF2-likenuclear transport factor 2 likePIC48S preinitiation complex