Phosphorylation/dephosphorylation of PTP-PEST at Serine 39 is crucial for cell migration.

We investigated the molecular details of the role of protein tyrosine phosphatase (PTP)-PEST in cell migration. PTP-PEST knockout mouse embryonic fibroblasts (KO MEFs) and MEF cells expressing a dominant-negative mutant of PTP-PEST showed significant suppression of cell migration compared to MEF cells expressing wild-type PTP-PEST (WT MEFs). Moreover, MEF cells harbouring a constitutively active mutant of PTP-PEST (S39A MEFs) showed a marked decrease in cell migration. In addition, MEF cells with no PTP-PEST or little PTP activity rapidly adhered to fibronectin and made many focal adhesions compared to WT MEF cells. In contrast, S39A MEF cells showed weak adhesion to fibronectin and formed a few focal adhesions. Furthermore, investigating the subcellular localization showed that Ser39-phosphorylated PTP-PEST was favourably situated in the adherent area of the pseudopodia. Therefore, we propose that suppression of PTP-PEST enzyme activity due to Ser39-phosphorylation in pseudopodia and at the leading edge of migrating cells induces rapid and good adherence to the extracellular matrix. Thus, suppression of PTP activity by Ser39-phosphorylation is critical for cell migration. Three amino acid substitutions in human PTP-PEST have been previously reported to alter PTP activity. These amino acid substitutions in mouse PTP-PEST altered the migration of MEF cells in a positive correlation.

Muscarinic receptor stimulation induces TASK1 channel endocytosis through a PKC-Pyk2-Src pathway in PC12 cells.

Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K+ 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCαactivates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners.

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways.

Protein phosphatase 1alpha associates with protein tyrosine phosphatase-PEST inducing dephosphorylation of phospho-serine 39.

Protein tyrosine phosphatase (PTP)-PEST is expressed in a wide variety of several cell types and is an efficient regulator of cell adhesion, spreading and migration. PTP-PEST-associating molecules are important in elucidating the function of PTP-PEST. Herein, we have identified protein phosphatase 1alpha (PP1alpha) as a novel PTP-PEST binding protein, and then we aimed to determine how PP1alpha contributes to the phosphorylation at Ser39 of PTP-PEST, whose phosphorylation suppresses PTP-PEST enzymatic activity. The HEK 293 cells overexpressing exogenous PTP-PEST were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39 PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated that the phosphorylation at Ser39 detected by anti-pS39-PEST Ab was dependent on TPA treatment and a significant inverse correlation between the PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive band intensity. The phosphorylation of Ser39 was suppressed by co-transfection of a plasmid encoding wild-type PP1alpha, but not by that of the dominant-negative PP1alpha mutant. Furthermore, TPA-induced phosphorylation could take place in PTP-PEST catalytic domain, but the phosphorylation of PTP-PEST catalytic domain could not be abrogated by co-transfection of a plasmid expressing wild-type PP1alpha. In conclusion, PP1alpha associates with the non-catalytic domain of PTP-PEST and regulates PTP activity via dephosphorylation of phospho-Ser39.

PICOT, protein kinase C theta-interacting protein, is a novel regulator of FcepsilonRI-mediated mast cell activation.

PICOT (PKC-interacting cousin of thioredoxin) consists of one thioredoxin homology domain in the N-terminal and two tandem PICOT homology domains in the C-terminal. PICOT specifically interacts with protein kinase C theta (PKC-theta) via its thioredoxin homology domain and acts as an important modulator of T cell receptor (TCR)-signaling. Using PICOT overexpressing rat basophilic leukemia cells (RBL-2H3), we evaluated the effect of PICOT overexpression on the FcepsilonRI-mediated signaling. In comparison to the control cells, introduction of PICOT to RBL-2H3 cells induced increased degranulation and the activation of NFAT and in the expression of IL-4 and TNF-alpha transcripts by FcepsilonRI-crosslinking, whereas no significant change was observed with the elevation of ERK1/2 and p38 MAP kinase phosphorylation and NF-kappaB activation by FcepsilonRI aggregation. More interesting was the exogenous PICOT overexpression in RBL-2H3 cells causing a large decrease in the elevation of JNK phosphorylation. PICOT-regulated FcepsilonRI-mediated signals in RBL-2H3 cells and acted as a positive regulator on IL-4 and TNF-alpha expression, NFAT and degranulation signal pathways and a negative regulator on a JNK signal pathway. Considering that PICOT has no enzymatic activity, the regulation of PICOT on FcepsilonRI-signaling may depend on PICOT-associated molecule(s).

Intracellular localization and domain organization of human TRIM41 proteins.

A human gene previously identified as a partial cDNA homologous to the gene of RET finger protein was characterized. Northern hybridization detected three messages of 3.3, 4.2, and 7.5 kb. The coding sequences of the more abundant of the three messages, the 4.2 and the 3.3 kb, were determined. The former encodes a 630 amino acid protein (TRIM41alpha) and the latter a 518 amino acid protein (TRIM41beta). Green fluorescent protein (GFP) fusions of full-length TRIM41alpha and TRIM41beta were both observed as speckles in the cytoplasm and the nucleus. The result was corroborated by Western analysis of cellular fractions. Results with GFP fusions of various segments of the TRIM41 proteins indicated that the nuclear transport of the proteins is mediated by an N-terminal segment common to both isoforms, but independent of a classical nuclear localization signal sequence.

Leupaxin binds to PEST domain tyrosine phosphatase PEP.

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes.

Gene expression profile in the livers of rats orally administered ethinylestradiol for 28 days using a microarray technique.

To identify genes showing responses to estrogen exposure in the livers of animals in a repeated oral dose toxicity study, dose-dependent gene expression profiles were analyzed using high-density oligonucleotide microarrays in Sprague-Dawley rats of both sexes administered ethinylestradiol (EE) for 28 days at concentrations of 0, 0.01, 0.1, and 1.0 ppm in the diet. Among 3776 genes examined, examples showing increased expression on EE-treatment were detected predominantly in females. Genes showing dose-dependent up-regulation with greater than five-fold change at 1.0 ppm from the control levels were found to, respectively, number 4 in males, and 24 in females. Most of the latter exhibited relatively high basal expression as well as low variability, and many exhibited clear dose-dependence. Genes showing dose-dependent down-regulation were rather few, and many of those affected exhibited relatively low expression levels with large variation between animals, like genes showing dose-unrelated expression patterns in both sexes or dose-dependent up-regulation in males. Considering that detection of changes in endocrine-linked organs and estrous cyclicity is only possible at the high dose of 1.0 ppm, up-regulation of genes dose-dependently in females provides a sensitive tool to detect estrogenic effects in the rat liver in the framework of the 28-day toxicity study.

Characterization of two isoforms of a human DnaJ homologue, HSJ2.

Two cDNA forms were characterized for a human dnaJ homologue, HSJ2. Nucleotide sequencing showed that the gene product HSJ2 was longer than previously reported, extending its homology to other human DnaJ paralogues, and that the two cDNAs encoded two proteins as a result of alternative splicing. The products were 326 amino acids (designated as HSJ2a) and 241 amino acids (HSJ2b) in length, sharing the N-terminal 231 amino acids including the DnaJ homology region. When fused to green fluorescent protein and expressed in HeLa cells, HSJ2a was found to be localized to the nucleus, indicating that HSJ2a is a nuclear co-chaperone. HSJ2b, however, was observed throughout the cell, consistent with the elimination of a putative nuclear localization signal sequence as a result of the alternative splicing.