MURINE PROTEIN-TYROSINE PHOSPHATASE-PEST, A STABLE CYTOSOLIC PROTEIN-TYROSINE-PHOSPHATASE

We have isolated the murine cDNA homologue of the human protein tyrosi ne phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic k idney library. The cDNA isolated has a single open reading frame predi cting a protein of 775 amino acids. When expressed in vitro as a gluta thione S-transferase fusion protein, the catalytic domain (residues 1- 453) shows intrinsic phosphatase activity. Reverse transcriptase PCR a nd Northern-blot analysis show that MPTP-PEST mRNA is expressed throug hout murine development. Indirect immunofluorescence in COS-1 cells ag ainst a heterologous epitope tag attached to the N-terminus of MPTP-PE ST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cy tosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in pr oline, glutamate, serine and threonine, otherwise known as PEST sequen ces. These are characteristic of proteins that display very short intr acellular half-lives. Despite the presence of these motifs, pulse-chas e labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.