PRLs (phosphatases of regenerating liver) are frequently overexpressed in human cancers and are prognostic markers of poor survival. Despite their potential as therapeutic targets, their mechanism of action is not understood in part due to their weak enzymatic activity. Previous studies revealed that PRLs interact with CNNM ion transporters and prevent CNNM4-dependent Mg 2+ transport, which is important for energy metabolism and tumor progression. Here, we report that PRL-CNNM complex formation is regulated by the formation of phosphocysteine. We show that cysteine in the PRL catalytic site is endogenously phosphorylated as part of the catalytic cycle and that phosphocysteine levels change in response to Mg
The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. Here, we characterize the interactions between the CBS-pair (Bateman) domain of CNNM3 and either PRL2 or PRL3 using X-ray crystallography, isothermal titration calorimetry, and activity assays. We report four new crystal structures of PRL proteins bound to the CNNM3 CBS-pair domain that reveal the effects of cysteine disulphide formation and nucleotide binding on complex formation. We use comprehensive mutagenesis of the PRL3 catalytic site to quantify the importance of different PRL amino acids, including cysteine 104, leucine 108, and arginine 110, for CNNM binding and phosphatase activity. We show the PRL3 R138E mutant is selectively deficient in CNNM3 binding with the potential to distinguish between the downstream effects of phosphatase and CNNM-binding activities in vivo. Through a novel activity assay, we show that PRL3 has magnesium-sensitive phosphatase activity with ATP and other nucleotides. Our results identify a strong correlation between phosphatase activity and CNNM binding and support the contention that PRL function as pseudophosphatases regulated by chemical modifications of their catalytic cysteine.
PDB Reference: third catalytic domain of ERp46, 3uvt.Protein disulfide isomerases are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum. Here, the crystal structure of the third catalytic domain of protein disulfide isomerase ERp46 (also known as protein disulfide isomerase A5 and TXNDC5) was determined to 2.0 Å resolution. The structure shows a typical thioredoxin-like fold, but also identifies regions of high structural variability. In particular, the loop between helix 2 and strand 3 adopts strikingly different conformations among the five chains of the asymmetric unit. Cys381 and Cys388 form a structural disulfide and its absence in one of the molecules leads to dramatic conformational changes. The tryptophan residue Trp349 of this molecule inserts into the cavity formed by helices 1 and 3 of a neighbouring molecule, potentially mimicking the interactions of ERp46 with misfolded substrates.
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The phosphatases of regenerating liver (PRLs) are highly over-expressed in metastatic cancers yet their mechanism of action is poorly understood. The PRL family belongs to the PTP (protein tyrosine phosphatase) superfamily and is comprised of three closely related proteins: PRL1, PRL2, and PRL3. They all contain a C-terminal prenylation site and a single catalytic domain of roughly 170 amino acids. Like other members of the family of protein tyrosine phosphatases, their phosphatase activity occurs through a two-step catalytic cycle involving the transient phosphorylation of a catalytic cysteine residue. In PRL phosphatases, this intermediate is extremely long-lived leading to the accumulation of a cysteine-phosphorylated form of the enzyme both in vitro and in cells [1,2]. While a number of different cellular substrates have been proposed, there is no consensus about their physiological substrate due, in part, to their slow rate of overall catalysis. The catalytic cysteine of PRLs readily forms a disulfide with the adjacent cysteine residue, which further decreases their effectiveness in dephosphorylating physiological substrates [1]. Recently, CNNM proteins, a family of membrane proteins involved in magnesium homeostasis, were identified as PRL-binding partners [3]. Disruption of the PRL-CNNM interaction promotes tumor formation and invasiveness in animal and cellular models, strongly suggesting that the physiological function of PRLs is to regulate CNNM magnesium transport. Here, we determined five crystal structures of PRL3 or PRL2 bound to the CBS-pair domain of CNNM3. In the structures, the CBS-pair domain is present as a dimer in the head-to-head arrangement that is typical for other CBS-pair domains. The CNNM3 CBS-pair domain contains a long loop that extends away from the dimerization interface and contacts the PRL catalytic site. The side chain of aspartic acid 426 sits in the pocket formed by the phosphatase P-loop and WPFDD motif and likely mimics the negatively charged phosphate of a bound substrate. Mutagenesis showed that Asp426 of CNNM3 is required for high affinity binding, suggesting that the CNNM CBS-pair domain might act as a pseudo-substrate. Addition of the CBS-pair domain inhibited phosphatase activity and CNNM3 binding was blocked by phosphorylation of the PRL active site cysteine. In addition to the many polar contacts, Pro427 and Tyr429 provide a hydrophobic surface that contacts Leu105 of PRL2 (Leu108 of PRL3). The structures reveal why disulfide formation dramatically decreases binding affinity and confirm that all three PRLs bind to CNNMs. We used isothermal titration calorimetry (ITC) experiments and extensive mutagenesis to probe the importance of PRL residues for CNNM binding. Comparison of binding activity and in vitro phosphatase activity shows that they are strongly correlated with the notable exception of the PRL3 R138E mutant which showed weak CNNM3 binding but normal phosphatase activity. These results support the hypothesis that PRLs function as pseudophosphatases in regulat...
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