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.

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Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.

Background: Protein phosphorylation and dephosphorylation is an integral component of many cellular signaling pathways and regulatory mechanisms. Phosphatases are enzymes that catalyze the removal of phosphate groups from proteins. The phosphatases of regenerating liver (PRLs) are a family of phosphatases which have been correlated with cancer development and metastasis. However, they appear to have weak phosphatase activity and little is known about their physiological substrates. This review discusses PRL from a structural and functional perspective, including recent findings on its interaction with another family of proteins, cyclin M (CNNM). Methods: Articles were obtained from the scientific literature using databases like PubMed and McGill University’s open access institutional repository. This paper specifically focuses on those articles that provided an overview of phosphatases, PRLs, CNNMs, and structural and functional studies of PRLs and CNNMs. In total, 40 articles were selected for the purpose of this review. Summary: Although PRLs retain many of the structural features of other protein tyrosine phosphatases (PTPs) including the phosphatase catalytic motif and regulation via oxidation, other structural features such as mutation of a conserved serine/threonine residue to alanine in the active site disfavor catalytic activity. Moreover, PRL interaction with CNNM appears to be responsible for its oncogenic potential, yet this inter- action does not appear to require PRL phosphatase activity. Thus, PRL may be best classified as a pseudo-phosphatase, which are phosphatase-like proteins that are structurally similar to phosphatases but have acquired a dominant function that does not require phosphatase activity.