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Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase
A Charest1, J Wagner, S H Shen
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abstract:
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.
Insights
Researchers identified the mouse homolog of human PTP-PEST (MPTP-PEST), a protein tyrosine phosphatase. Despite containing PEST sequences, MPTP-PEST exhibits a longer-than-expected cellular half-life, suggesting unique regulatory mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Protein tyrosine phosphatases (PTPs) play critical roles in cellular signaling pathways.
- The human PTP-PEST protein is known to be involved in regulating cell proliferation and survival.
- Understanding the function and regulation of PTP-PEST homologues in other species can provide insights into conserved biological processes.
Purpose of the Study:
- To isolate and characterize the murine homolog of human PTP-PEST (MPTP-PEST).
- To investigate the expression pattern, cellular localization, and enzymatic activity of MPTP-PEST.
- To determine the intracellular half-life of MPTP-PEST and its relationship with PEST sequences.
Main Methods:
- Isolation of murine cDNA from an embryonic kidney library.
- In vitro expression and characterization of the catalytic domain.
- Reverse transcriptase PCR and Northern-blot analysis for mRNA expression.
- Indirect immunofluorescence, cellular fractionation, and Western-blot for protein localization.
- Pulse-chase labeling experiments to determine protein half-life.
Main Results:
- The murine cDNA homologue, MPTP-PEST, was isolated, predicting a 775-amino acid protein.
- The catalytic domain demonstrated intrinsic phosphatase activity.
- MPTP-PEST mRNA was detected throughout murine development.
- MPTP-PEST was identified as a 112 kDa free cytosolic protein.
- Despite containing PEST sequences, MPTP-PEST exhibited a half-life exceeding 4 hours.
Conclusions:
- MPTP-PEST is a functional protein tyrosine phosphatase expressed during murine development.
- The protein localizes to the cytosol and possesses catalytic activity.
- The unexpected stability of MPTP-PEST, despite the presence of degradation-associated PEST motifs, indicates complex post-translational regulation.