Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2

Background - Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. // Methodology/Principal Findings - Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C2C12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C2C12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. // Conclusions/Significance - Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation

Increased corticosteroid binding capacity of plasma albumin but not of corticosteroid-binding globulin caused by ACTH-induced changes in free fatty acid concentrations in snowshoe hares and rabbits

Free fatty acids (FFAs) are rapidly mobilized by ACTH and have been shown to be potent endogenous modulators of steroid–protein interactions. We increased FFA in lagomorphs by ACTH and then separated the transient increase in glucocorticoid binding capacity of plasma into that accounted for by changes in binding to albumin and to corticosteroid-binding globulin (CBG). Sequential injections of dexamethasone and ACTH into both snowshoe hares and laboratory rabbits resulted in the rapid mobilization of FFA only after the ACTH injection. The maximum corticosteroid binding capacity increase paralleled that of the FFA increase in both species. In rabbits, CBG levels remained constant over the duration of the experiment. Corticosterone binding by rabbit albumin increased in a dose-dependent fashion in response to increases in FFA (oleic and linoleic acid) concentrations. Finally, by stimulating FFA release in snowshoe hares with ACTH and separating the increase in corticosteroid binding capacity through selective denaturing of CBG by heat, we determined that the increase in plasma binding capacity was a response to changes in binding by albumin, not CBG. Thus FFA released in response to stressors in lagomorphs may effect short-term increases in steroid binding

A novel cell lysis approach reveals that caspase-2 rapidly translocates from the nucleus to the cytoplasm in response to apoptotic stimuli.

Unlike other caspases, caspase-2 appears to be a nuclear protein although immunocytochemical studies have suggested that it may also be localized to the cytosol and golgi. Where and how caspase-2 is activated in response to apoptotic signals is not clear. Earlier immunocytochemistry studies suggest that caspase-2 is activated in the nucleus and through cleavage of BID leads to increased mitochondrial permeability. More recent studies using bimolecular fluorescence complementation found that caspase-2 oligomerization that leads to activation only occurs in the cytoplasm. Thus, apoptotic signals may lead to activation of caspase-2 which may already reside in the cytoplasm or lead to release of nuclear caspase-2 to the extra-nuclear cytoplasmic compartment. It has not been possible to study release of nuclear caspase-2 to the cytoplasm by cell fractionation studies since cell lysis is known to release nuclear caspase-2 to the extra-nuclear fraction. This is similar to what is known about unliganded nuclear estrogen receptor-α (ERα ) when cells are disrupted. In this study we found that pre-treatment of cells with N-ethylmaleimide (NEM), which alkylates cysteine thiol groups in proteins, completely prevents redistribution of caspase-2 and ERα from the nucleus to the extra-nuclear fraction when cells are lysed. Using this approach we provide evidence that apoptotic signals rapidly leads to a shift of caspase-2 from the nucleus to the extra-nuclear fraction, which precedes the detection of apoptosis. These findings are consistent with a model where apoptotic signals lead to a rapid shift of caspase-2 from the nucleus to the cytoplasm where activation occurs

Etoposide incubation leads to rapid accumulation of caspase-2 in the extra-nuclear fraction of lysed cells.

<p>The results show the accumulation of caspase-2 in the extra-nuclear fraction (designated as lysate) after treating cells for 15 min to 3 h with 100 uM etoposide. After etoposide incubation the medium was replaced with serum free medium containing 20 mM NEM for 10 min before lysis.</p

Effect of NEM on the cell distribution of wild-type and mutant caspase- 2.

<p>Shown is the distribution of wild-type GFP-Caspase-2 (GFP-C2-WT) (A), and mutant GFP-caspase-2 (GFP-C2-MUT) (B) between nuclear and extra-nuclear fractions of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis. Also shown in both panels is the distribution of endogenously expressed caspase-2 in response to NEM pre-treatment in the same cells. The numbers on the right of the panels reflect the gel migration of the 40 kDa and 70 kDa protein markers.</p

H₂O₂ incubation leads to rapid accumulation of caspase-2 in the extra-nuclear fraction of lysed cells.

<p>(A) Caspase-2 in the extra-nuclear fraction (designated as lysate) after treating cells for 1 and 3 h with concentrations of H₂O₂ known to induce apoptosis. (B) Caspase-2 levels in the extra-nuclear fraction after treatment with 20 mM H₂O₂ for 5 or 15 min. After H₂O₂ incubation the medium was replaced with serum free medium containing 20 mM NEM for 10 min before lysis.</p

MATHEMATICAL MODEL OF THE FUNCTIONING PROCESS OF A RUBBER BUSHING ON THE HOOKE AND SAINT-VENANT ELEMENTS’ BASIS

Introduction. Rubber bushings are important parts of the spring systems of modern vehicles. The properties determine not only the comfort of car movement, but also affect the elastic suspension characteristics. When a bushing is deformed, linear sections appear in characteristics. It is advisable to describe such characteristics using a mathematical model based on the classical elements of Hooke and Saint-Venant. The paper presents a mathematical description of the bushing simulation functioning results, accuracy of approach, areas of application of the mathematical model.Materials and methods. The initial data was the experimental characteristic of a cylindrical automobile rubber bushing, obtained in harmonic mode at the 0.03 to 51 Hz frequency and the 0.4 to 10 mm amplitude. The force balance of the two Hooke elements and one Saint-Venant element interacting with each other determined the mathematical model description. The authors carried out the calculations using numerical and optimization methods.Results. As a result, the authors determined functions characterizing the change in the parameters of the Hooke and Saint-Venant elements from the rubber bushings’ deformation amplitude. Moreover, the authors calculated power characteristics in the form of dependences of the rubber bushing effort and also found quantitative indicators of the reliability of the experimental data approximation by the developed mathematical model.Discussion and conclusions. The analysis of the operating modes shows the possibility of the model application to describe the rubber bushing functioning in a stationary harmonic mode with small and medium strain amplitudes. The simulation results of the Hooke’s and Saint-Venant’s parameters reveals the theoretical prerequisites for the possibility of using the model to calculate the bushing force in an unsteady mode.The authors have read and approved the final manuscript. Financial transparency: the authors have no financial interest in the presented materials or methods. There is no conflict of interest

Effect of NEM and IAA concentrations on endogenous nuclear caspase-2 levels.

<p>(A) Cells were pretreated with the indicated concentrations of NEM for 10 min before lysis. (B) Effect of NEM concentrations on nuclear caspase-2 levels. Cells were pre-treated with NEM as indicated for 1 h before lysis. (C) Effect of 10 min or 1 h pre-treatment with the indicated concentrations of Iodoacetic Acid (IAA) or NEM on caspase-2 levels in the nuclear fraction.</p