HDPTP · Molecular analysis of His-domain protein tyrosine phosphatase, a putative tumour suppressor

Final Activity Report Summary - HDPTP (Molecular analysis of His-domain protein tyrosine phosphatase, a putative tumour suppressor)

Tyrosine phosphorylation participates in a wide array of signal transduction pathways essential for proper control of several cellular activities, including motility, metabolism, differentiation, cell growth and proliferation. Thus, tyrosine kinases and tyrosine phosphatases are important regulators of these signal transduction pathways. His-domain-containing protein tyrosine phosphatase (HD-PTP) is classified as a cytosolic protein tyrosine phosphatase (EC 3.1.3.48). The human protein has several structural domains: a Bro1 like domain and a coiled-coil domain toward the N-terminal end, a histidine domain that is a proline rich domain unique to this protein, a protein tyrosine phosphatase domain with unique key residues in its catalytic structural motifs, and two PEST motifs at its C-terminal end. In our study, using a yeast two-hybrid screen, we found new interactors of HD-PTP: GRB2-related adaptor protein 2, RNA binding motif protein 35B, growth factor receptor bound protein 2, and IgA2. Further studies are underway to confirm that these interactions have indeed functional relevance. An important discovery made during the project was the identification of the HD-PTP structural domain responsible for the protein association with intracellular vesicles. When overexpressed in HEK 293 and HeLe cells, HD-PTP appears to be dispersed throughout the cytosol while at the same time concentrated to some vesicular structures throughout the cytosol. This suggests that HD-PTP might be targeted to intracellular vesicles under certain physiological conditions. We ectopically expressed a series of deletion mutants in both HEK293 and HeLe cells and followed their localisation by fluorescence microscopy. Thus, we found that the coiled-coil domain of HD-PTP corresponding to the 403-704 residues of the amino acid backbone is essential for this vesicular localisation. We also found out that the Bro1 domain (1-403 amino acid residues) and the central Proline-rich domain (705-1150 residues) were not involved in targeting the protein to vesicles. We initiated immunocytochemistry experiments with several organelle markers to identify the HD-PTP-bound vesicles. Thus far we found that these vesicles are not the early endosomes, nor the lysobisphosphatidic acid-enriched vesicles of the multivesicular bodies (MVBs). However, the latter observation does not rule out the potential of HD-PTP association with the external MVB membrane. Our data have shown that the catalytic domain of HD-PTP does not hydrolyse para-nitrophenyl phosphate or the phosphatidylinositol phosphates in vitro. This might be explained by the particular aminoacid residues present in the structural motifs of the catalytic domain. Interestingly, in our in vitro protein pull-down experiments, we isolated several phosphorylated proteins that bound to the catalytic domain. We observed also that the substitution of the Cys from the consensus catalytic site of protein tyrosine phosphatases did not change the binding affinity of this domain for phosphorylated proteins. Further experiments are on going to try to identify these interactors. Lastly, but not least, we were able to partially clone the zebrafish (Danio rerio) cDNA. By Northern blot analysis of total RNA isolated from adult fish we determined that the apparent molecular size of zebrafish HD-PTP transcript is around 7 kb. By reverse transcription-polymerase chain reaction we found that the messenger RNA for the protein is present in unfertilised eggs and also in every developmental stage tested (cleavage period: 2 cell, 64 cell, gastrula period: shield, 75% epiboly, segmentation period: 4 somite, 17 somite, hatching period: 48 h). Our findings on the early expression of the gene are the first data regarding the role of HD-PTP in development and they suggest that this protein might be essential for different developmental processes. Our hypothesis will be tested during the third year of this project.

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