ABSTRACT
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.
Subject(s)
Fibronectins , Serine , Animals , Mice , Humans , Phosphorylation , Fibronectins/metabolism , Fibroblasts/metabolism , Cell MovementABSTRACT
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.
Subject(s)
Mast Cells/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 12/immunology , Receptors, IgE/immunology , Animals , Cell Degranulation/genetics , Cell Degranulation/immunology , Cell Line , HEK293 Cells , Humans , Inflammation/immunology , Interleukin-13/biosynthesis , Interleukin-13/genetics , Interleukin-4/biosynthesis , Interleukin-4/genetics , Phosphorylation , Protein Binding/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 12/biosynthesis , Protein Tyrosine Phosphatase, Non-Receptor Type 12/genetics , Rats , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
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.
