Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase.
We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
Charest
A
A
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Wagner
J
J
Shen
S H
SH
Tremblay
M L
ML
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008969:Molecular Sequence Data; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping
D017931:DNA Primers; D012333:RNA, Messenger; C516738:PTPN12 protein, human; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases; C516739:Ptpn12 protein, mouse