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The MAP-kinase ERK2 is a specific substrate of the protein tyrosine phosphatase HePTP
1DNAX Research Institute, 901 California Avenue, Palo Alto, California, CA 94304, USA.
Abstract:
HePTP is a tyrosine specific protein phosphatase that is strongly expressed in activated T-cells. It was recently demonstrated that in transfected T-cells HePTP impairs TCR-mediated activation of the MAP-kinase family members ERK2 and p38 and it was suggested that both ERK and p38 MAP-kinases are substrates of HePTP. The HePTP gene has been mapped to human chromosome 1q32.1. Abnormalities in this region are frequently found in various hematopoietic malignancies. HePTP is highly expressed in acute myeloid leukemia and its expression in fibroblasts resulted in transformation. To address a possible involvement of HePTP in hematopoietic malignancies we sought to identify HePTP substrate(s) in leukemic cells. Using substrate trapping mutants we have identified the MAP-kinase ERK2 as a specific target of HePTP in the myelogenous leukemia cell line K562. Tyrosine phosphorylated ERK2, but not ERK1, p38, or JNK1, efficiently bound to catalytically inactive HePTP mutants in which the active site cysteine (HePTP-C/S) or the conserved aspartic acid residue (HePTP-D/A) had been exchanged for serine and alanine, respectively. Moreover, the interaction of ERK2 with HePTP trapping mutants was dependent on ERK2 tyrosine phosphorylation, indicating that HePTP is specifically targeted to activated ERK2. Using a deletion mutant of HePTP (HePTP-dLD), in which 14 amino acid residues within the N-terminus are missing, we show that regions outside the catalytic domain are also required for the interaction. Furthermore, overexpression of HePTP in K562 cells and fibroblasts interfered with PMA or growth factor induced MAP-kinase activation and HePTP efficiently dephosphorylated active ERK2 on the tyrosine residue in the activation loop in vitro. Together, these data identify ERK2 as a specific and direct target of HePTP and are consistent with a model in which HePTP negatively regulates ERK2 activity as part of a feedback mechanism. Oncogene (2000) 19, 858 - 869.
Insights
HePTP phosphatase directly targets and dephosphorylates the MAP-kinase ERK2 in leukemia cells. This interaction suggests HePTP negatively regulates ERK2 activity, potentially playing a role in hematopoietic malignancies.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- HePTP (Hepatoma-derived tyrosine phosphatase) is a tyrosine phosphatase highly expressed in activated T-cells.
- HePTP gene is located on chromosome 1q32.1, a region associated with hematopoietic malignancies.
- HePTP overexpression in fibroblasts causes transformation, suggesting a role in cancer.
Purpose of the Study:
- To identify substrates of HePTP in leukemic cells.
- To investigate the potential involvement of HePTP in hematopoietic malignancies.
- To elucidate the regulatory mechanism of HePTP on MAP-kinases.
Main Methods:
- Utilized substrate trapping mutants of HePTP (HePTP-C/S and HePTP-D/A) to identify interacting proteins.
- Employed myelogenous leukemia cell line K562 for experiments.
- Used a deletion mutant (HePTP-dLD) to analyze functional domains.
- Performed in vitro dephosphorylation assays.
Main Results:
- Identified MAP-kinase ERK2 as a specific substrate of HePTP in K562 cells.
- Demonstrated that HePTP specifically binds to tyrosine-phosphorylated ERK2.
- Showed that regions outside HePTP's catalytic domain are necessary for ERK2 interaction.
- Confirmed HePTP dephosphorylates active ERK2 in vitro, inhibiting MAP-kinase activation.
Conclusions:
- ERK2 is a direct and specific target of HePTP.
- HePTP negatively regulates ERK2 activity, likely through a feedback mechanism.
- These findings support a role for HePTP in hematopoietic malignancies.