Structure and Biochemical Properties of PRL-1, a Phosphatase Implicated in Cell Growth, Differentiation, and Tumor Invasion^,

Abstract

The PRL (phosphatase of regenerating liver) phosphatases constitute a novel class of small, prenylated phosphatases that are implicated in promoting cell growth, differentiation, and tumor invasion, and represent attractive targets for anticancer therapy. Here we describe the crystal structures of native PRL-1 as well as the catalytically inactive mutant PRL-1/C104S in complex with sulfate. PRL-1 exists as a trimer in the crystalline state, burying 1140 A2 of accessible surface area at each dimer interface. Trimerization creates a large, bipartite membrane-binding surface in which the exposed C-terminal basic residues could cooperate with the adjacent prenylation group to anchor PRL-1 on the acidic inner membrane. Structural and kinetic analyses place PRL-1 in the family of dual specificity phopsphatases with closest structural similarity to the Cdc14 phosphatase and provide a molecular basis for catalytic activation of the PRL phosphatases. Finally, native PRL-1 is crystallized in an oxidized form in which a disulfide is formed between the active site Cys104 and a neighboring residue Cys49, which blocks both substrate binding and catalysis. Biochemical studies in solution and in the cell support a potential regulatory role of this intramolecular disulfide bond formation in response to reactive oxygen species such as H2O2.

Characterization of the Protein Tyrosine Phosphatase LmPRL-1 Secreted by Leishmania major via the Exosome Pathway

“…The analysis of their sequences also revealed a strong similarity to the mammalian PRL phosphatase family, as illustrated by the conservation of the classical catalytic site (C-X 5 -R), a regulatory cysteine at the N terminus, and a farnesylation site at the C terminus. The suspected homology was confirmed by the in vitro biochemical characterization of His 6 -LmPRL-1 and comparison with the known characteristics of the human PRL-1 (50,53). First, the catalytic constants (K m ) of His 6 -LmPRL-1 using the DiFMUP substrate (9.32 ϫ 10 Ϫ6 M) was in line with the previously measured value for its human homologue, PRL-1 (4.6 ϫ 10 Ϫ6 M) (50).…”

“…The suspected homology was confirmed by the in vitro biochemical characterization of His 6 -LmPRL-1 and comparison with the known characteristics of the human PRL-1 (50,53). First, the catalytic constants (K m ) of His 6 -LmPRL-1 using the DiFMUP substrate (9.32 ϫ 10 Ϫ6 M) was in line with the previously measured value for its human homologue, PRL-1 (4.6 ϫ 10 Ϫ6 M) (50). In addition, the activity of the phosphatase was shown to be strictly dependent on the reduction of a disulfide bridge formed between its catalytic cysteine and its regulatory cysteine.…”

“…1C). In PRL-1, this proximity between cysteines was previously described as essential for the regulation of catalytic activity, since the two residues could form an inactivating disulfide bond (50,51). Another important feature shared by all these phosphatases is the presence of a farnesylation site (C-A-A-X) at the C terminus of the protein, which suggests possible membrane localization for the Leishmania phosphatases (Fig.…”

“…2A). Since PRL-1 has higher affinity for the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), this substrate was chosen for the further catalytic study of His 6 -LmPRL-1 (50). The optimal pH for the activity of His 6 -LmPRL-1 turned out to be slightly acidic (pH 6.0) (Fig.…”

Structural basis for the glucan phosphatase activity of Starch Excess4

“…Many dual specificity phosphatases contain little more than a DSP domain and can be readily expressed in Escherichia coli (38)(39)(40)(41). The DSP of SEX4 cannot be independently expressed, and we hypothesized that the intimate association of the C-terminal domain with the DSP underlies this phenomenon.…”