Characterization, expression and functional aspects of a novel protein tyrosine phosphatase epsilon isoform.

This report describes the identification and characterization of a novel cytoplasmic isoform of human protein tyrosine phosphatase epsilon (PTPepsilon). The novel isoform, denoted cyt-PTPepsilonPD1, displays only the N-terminal catalytic, active phosphatase domain 1 (PD1) which is common in all known PTPepsilon isoforms. In addition, it contains a unique 132-residue long C-terminal end with no known motifs or homology to other characterized proteins. RNAse protection assay on isolated leucocyte subpopulations and selected cell lines demonstrated highest expression of cyt-PTPepsilonPD1 in monocytes. The mRNA-encoding cyt-PTPepsilonPD1 is detected as distinct transcript(s) by Northern blot analysis and is a result of alternative splicing. cyt-PTPepsilonPD1 shows similar cellular localization in transfected cells, both in the cytoplasm and nucleus, as has been previously described for cytoplasmic PTPepsilon isoform. Our previous data suggest that the expression of cytoplasmic PTPepsilon inhibits the mitogen-activated protein kinase cascade through the extracellular signal-regulated kinase 1 and 2 pathway. A similar functional role is also presented here for cyt-PTPepsilonPD1, supporting our previous data suggesting that the catalytic first PD of PTPepsilon is responsible for this inhibition.

Expression of human protein tyrosine phosphatase epsilon in leucocytes: a potential ERK pathway-regulating phosphatase.

The expression of protein tyrosine phosphatase epsilon (PTPepsilon) was studied in human tissues and blood cells. High mRNA expression was observed in peripheral blood leucocytes, particularly in monocytes and granulocytes which revealed at least four distinct transcripts. In lymphocytes, PTPepsilon expression was induced after 12-O-tetradecanoylphorbol-13-acetate (TPA) or antigen-receptor stimulation, indicating that PTPepsilon plays a role in the events taking place after antigen engagement. Previously, PTPepsilon has been shown to be involved in regulating voltage-gated potassium channel activity, insulin-receptor signalling and Janus kinase-signal transducers and activators of transcription (STAT) signalling. Transfection of cells with different PTPepsilon constructs and activator protein-1 reporter gene indicates that the catalytic activity of PTPepsilon is involved in the regulation of the mitogen-activated protein kinase cascade. In particular, the extracellular signal-regulated kinases (ERK1/2) were shown to be inhibited in both phosphorylation status and enzymatic activity after overexpression of PTPepsilon. Thus, PTPepsilon emerges as a phosphatase with a potential to regulate the ERK1/2 pathway either directly or indirectly through its catalytic activity.

A signal sequence trap based on cell enrichment using anti-CD19 antibody coated magnetic beads.

Precursors of many secreted and cell surface proteins contain NH2-terminal signal sequences that lead proteins into the endoplasmic reticulum and to the cell surface. Methods have been developed to clone and identify such proteins by trapping their NH2-terminal signal sequences, so called signal sequence traps. In this study we present an alternative and simplified signal sequence trap based on the combination of a novel vector construct expressing a cDNA library in fusion with a CD19 reporter gene, transfection in mammalian cells and selection of cells expressing trapped signal sequences using magnetic beads. Using this method we have isolated several known and novel factors with signal peptides.