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

The pseudophosphatase MK-STYX (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein) has been implicated in the stress response pathway. The expression of MK-STYX inhibits the assembly of stress granules, which are cytoplasmic storage sites for mRNA that form as a protective mechanism against stressors such as heat shock, UV irradiation and hypoxia. Furthermore, MK-STYX interacts with a key component of stress granules: G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1). Because G3BP-1 dephosphorylation at Ser149 induces stress granule assembly, we initially hypothesized that the inhibition of stress granules by MK-STYX was G3BP-1 phosphorylation-dependent. However, in the present study, using MK-STYX constructs and G3BP-1 phosphomimetic or nonphosphorylatable mutants, we show that MK-STYX inhibits stress granule formation independently of G3BP-1 phosphorylation at Ser149. The introduction of point mutations at the active site of MK-STYX that convert serine and phenylalanine to histidine and cysteine, respectively, is sufficient to generate an active enzyme. In separate experiments, we show that this active mutant, MK-STYXactive, has opposite effects to wild-type MK-STYK. Not only does MK-STYXactive induce stress granules, but also it has the capacity to dephosphorylate G3BP-1. Taken together, these results provide evidence that the pseudophosphatase MK-STYX plays a key role in the cellular response to stress

Collapse of the North American ice saddle 14,500 years ago caused widespread cooling and reduced ocean overturning circulation

Collapse of ice sheets can cause significant sea level rise and widespread climate change. We examine the climatic response to meltwater generated by the collapse of the Cordilleran-Laurentide ice saddle (North America) ~14.5 thousand years ago (ka) using a high-resolution drainage model coupled to an ocean-atmosphere-vegetation general circulation model. Equivalent to 7.26 m global mean sea level rise in 340 years, the meltwater caused a 6 sverdrup weakening of Atlantic Meridional Overturning Circulation (AMOC) and widespread Northern Hemisphere cooling of 1–5°C. The greatest cooling is in the Atlantic sector high latitudes during Boreal winter (by 5–10°C), but there is also strong summer warming of 1–3°C over eastern North America. Following recent suggestions that the saddle collapse was triggered by the Bølling warming event at ~14.7–14.5 ka, we conclude that this robust submillennial mechanism may have initiated the end of the warming and/or the Older Dryas cooling through a forced AMOC weakening

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

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(X-5)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

MK-STYX shifts the distribution of neurite outgrowth lengths.

<p>(<b>A</b>) The length of a neurite-like outgrowth of PC12 cells over-expressing GFP and MK-STYX was measured to demonstrate how cells were scored for outgrowths ≧20 µm. (<b>B</b>) PC12 cells over-expressing GFP and MK-STYX or pMT2 control plasmid for 24 hr were stimulated with 100 ng/ml NGF. Twenty-four hr post-stimulation cells were scored (n = 200) for outgrowth length. Cells were (<b>C</b>) not stimulated or (<b>D</b>) stimulated with 100 ng/ml of NGF. White arrows indicate “branching out” of primary neurites. (<b>E</b>) Cells were scored for neurite extensions ≧20 µm, and statistical analysis was performed using ANOVA (F_2,8 = 43.81, ***p<0.001).</p

Pseudophosphatase MK-STYX induces neurite extensions in PC12 cells.

<p>(<b>A</b>) Representative examples are presented to illustrate neurite outgrowths of PC12 cells over-expressing MK-STYX and GFP, MK-STYX_active, or pMT2 control plasmid and GFP. Cells were incubated 5 days. (<b>B</b>) Cells transfected with pEGFP, pMT2, or pMT2-FLAG-MK-STYX-FLAG plasmids were scored for neurite extensions ≥20 µm with a phase objective. Three replicate experiments were performed (n = 100 cells per experiment); the results are ± SEM. Statistical analysis was performed using ANOVA (F_2,8 = 76.10, ***p<0.0001).</p

MK-STYX induces neurites in the presence of MEK inhibitor (PD98059).

<p>Representative examples of PC12 cells over-expressing pMT2 and GFP, or MK-STYX and GFP, that were stimulated with 100 ng/ml NGF and (<b>A</b>) treated with, or (<b>B</b>) not treated with 50 µM PD98059. (<b>C</b>) Cells were scored for neurite extensions ≧20 µm. Statistical analysis was performed with t-tests for control group (pMT2 and pEGFP; **p<0.01) and experimental group (MK-STYX and pEGFP; **p<0.01).</p