The pseudophosphatase <scp>MK</scp>‐<scp>STYX</scp> inhibits stress granule assembly independently of Ser149 phosphorylation of G3<scp>BP</scp>‐1

Barr, Justinn E.; Munyikwa, Michelle; Frazier, Elizabeth A.; Hinton, Shantá D.

doi:10.1111/febs.12068

Cited by 26 publications

(25 citation statements)

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“…Mutations made in MK-STYX that restore this signature motif generate an active phosphatase (MK-STYX active ) [34] . Despite being catalytically inactive, MK-STYX plays a role in a number of important cellular pathways [34] – [36] , including inhibition of apoptosis and stress granule formation [34] , [35] , [37] . The functionality of MK-STYX is underexplored and still remains elusive; however, as with active MKP homologs, determining its interaction partners has helped elucidate some of the functions of MK-STYX [34] , [38] .…”

Section: Introductionmentioning

confidence: 99%

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The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

et al. 2014

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The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.

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Section: Introductionmentioning

confidence: 99%

“…We determined that MK-STYX inhibits stress granule formation [34] . Dephosphorylation of G3BP-1 at Ser149 is critical for stress granule formation [39] ; however, MK-STYX's interaction with G3BP-1 and its effect on the stress granule pathway is independent of the phosphorylation status of Ser 149 [37] .…”

Section: Introductionmentioning

confidence: 99%

The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

et al. 2014

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“…Hinton et al showed that overexpressed pseudo-phosphatase MK-STYX interacts with G3BP1 and inhibits the assembly of SGs, and first hypothesized that MK-STYX inhibition of SG assembly is G3BP-S149 dependent (Hinton et al, 2010), but subsequently showed that SG assembly is independent of S149 phosphorylation (Barr et al, 2013), using the original phosphomimetic (S149E) and non-phosphorylatable (S149A) mutants. Surprisingly, overexpression of mutationally-activated MK-STYX shows opposite effects; it induces rather than blocks SGs as does the native, enzymatically-dead version, and is capable of de-phosphorylating G3BP (Barr et al, 2013). Considering the SG-inhibitory effects of the S99P mutation in the S149E construct, it is possible that a clean S149E construct co-expressed with MK-STYX (native) would not have the same effects, as it allows the formation of SGs to the same extent as WT (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Here we report that there are additional mutations in the original pEGFP-C1-G3BP1-S149A and S149E constructs (Tourriere et al, 2003) that were generously shared and widely disseminated. These constructs were used in our study (Kedersha et al, 2016) and many other studies (Barr et al, 2013; Jedrusik-Bode et al, 2013; Kedersha et al, 2016; Kwon et al, 2007; Sahoo et al, 2018; Sahoo et al, 2012; Szaflarski et al, 2016). We have now identified second mutations within the NTF2-like domains of the original constructs.…”

Section: Introductionmentioning

confidence: 99%

Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly

Panas

Kedersha

Schulte

et al. 2019

Preprint

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34Stress granules (SGs) are cytoplasmic, non-membranous RNA/protein structures that assemble 35 in response to environmental stress. G3BP is a critical SG-nucleating protein, and its ability to 36 regulate SGs has been reported to be regulated by serine 149 phosphorylation. We now report 37 that the constructs engineered to contain non-phosphorylatable and phosphomimetic (G3BP1-38 S149A and G3BP1-S149E, respectively) mutations used in many studies include additional 39 unintended mutations (A54T/S149A and S99P/S149E) one of which (S99P) is responsible for the 40 effects on SG assembly attributed to S149E. Specifically, the S99P mutation alone reduces SG 41 nucleation and impairs the ability to rescue SG assembly in G3BP1/2 U2OS KO cells, 42 challenging the widely-stated conclusion that de-phosphorylation of serine 149 in G3BP1 43 promotes SG assembly. We used comparative mass spectrometry analysis of both (1) ectopically 44 expressed GFP-G3BP1 in G3BP1/2 U2OS KO and (2) endogenous G3BP1 in wild-type U2OS, 45 with and without sodium arsenite treatment, in an attempt to reproduce earlier findings, but 46 found no significant changes in S149 phosphorylation that correlate with arsenite-induced SG 47 formation.

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“…This pseudophosphatase has the potential to affect different proteins/pathways, including ERKs (Reiterer et al, 2013), PTPMP1 (Niemi et al, 2014) and Ras signaling through interaction with G3BP‐1 (Barr et al, 2013). Downregulation of Styx caused an increase in 4E‐BP1 phosphorylation at T37/46, similarly to what was observed after APP depletion.…”

Section: Discussionmentioning

confidence: 99%

Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

Sobol¹,

Galluzzo²,

Liang³

et al. 2015

Journal Cellular Physiology

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Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. 230: 1064–1074, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

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The pseudophosphatase MK‐STYX inhibits stress granule assembly independently of Ser149 phosphorylation of G3BP‐1

Cited by 26 publications

References 32 publications

The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly

Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

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