Purification and characterization of protein tyrosine phosphatase PTP‐MEG2

Abstract

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.

Homotypic Secretory Vesicle Fusion Induced by the Protein Tyrosine Phosphatase MEG2 Depends on Polyphosphoinositides in T Cells

“…When the lipid is present, PTP-MEG2 dephosphorylates one or several substrates involved in secretory vesicle fusion and/or homeostasis. This model is supported by the recent finding that PTP-MEG2 lacking its Sec14p homology domain has a considerably higher activity than the full-length enzyme (42). Thus, the Sec14p homology domain appears to be involved in intramolecular regulation of the catalytic activity of PTP-MEG2.…”

The Sec14 Homology Domain Regulates the Cellular Distribution and Transforming Activity of the Rho-specific Guanine Nucleotide Exchange Factor Dbs

“…Although the function and lipid binding specificity of the domain in the context of other RhoGEFs has yet to be determined, recent studies of the Sec14 homology domain of the protein phosphatase MEG2 demonstrated that this domain is responsible for regulation of the MEG2 catalytic activity (61) and that it modulates intracellular localization of the protein though specific binding to phosphatidylserine (62). Our demonstration that the Sec14 domain of Dbs has similar properties suggests a conserved function for these sequences in the context of mammalian proteins.…”

PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells

“…However, with a nonfusion protein purified from an adenovirus expression system, we failed to show such an activation. 23 Instead, our recent studies demonstrated a specific binding of PTP-MEG2 with phosphatidylserine through the putative lipidbinding domain but the lipid does not activate the enzyme in vitro. 36 What activates PTP-MEG2 PV ECFCs remains to be identified.…”

“…Recent studies showed that removal of the N-terminal domain significantly activated the enzyme, suggesting its regulatory role. 23,32 Furthermore, PTP-MEG2 has been shown to be partially localized in intracellular membranes at the secretory vesicles. 32,33 In this study, we have demonstrated that PTP-MEG2 is present in both cytosol and membrane fractions of ECFCs.…”

“…Both mutants are catalytically inactive. 23 The pMSCV-IRES-GFP vector has an internal ribosome entry site (IRES) that enables expression of target gene and green fluorescence protein (GFP) as separated proteins from a single transcript initiating in the 5Ј long-terminal repeat. GFP is used as a marker to indicate the efficiency of gene transfer.…”