PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3

Abstract

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.

Physiological and oncogenic roles of the PRL phosphatases

“…This model was further supported by voltage-clamp studies using a CNNM3 mutant that is unable to bind the PRLs [189]. This influx mechanism is consistent with the function of CNNM2, which increases the uptake of 25 Mg isotope and where the T568I mutation found in patients was linked with hypomagnesemia caused by impaired magnesium uptake [213,214]. Funato et al identified similar binding of PRL-3 and CNNM4 interaction in colorectal cancer [184].…”

“…Such an increase in intracellular magnesium will lead to a higher abundance of Mg‐ATP in the cell, which can bind allosterically to the PRL/CNNM complex and maintain the flat conformation . It was proposed that stabilization of the complex is dependent on the conserved aspartic acid residue present on the tip of the CBS loop of CNNMs confirming the initial finding by Kostantin et al . .…”

“…An independent report by a second group also showed this unexpected molecular association [184]. This interaction was further validated by structural studies, which characterized the interactions between both proteins [23,25,185]. The CNNMs, previously known as ancient conserved domain protein (ACDP), are found expressed in mammals and are evolutionary conserved from unicellular to multicellular organisms [186,187].…”

“…Such an increase in intracellular magnesium will lead to a higher abundance of Mg-ATP in the cell, which can bind allosterically to the PRL/CNNM complex and maintain the flat conformation [185]. It was proposed that stabilization of the complex is dependent on the conserved aspartic acid residue present on the tip of the CBS loop of CNNMs [25,185,189] confirming the initial finding by Kostantin et al [182]. Moreover, this aspartic acid plays a substrate analog-like role on the active site of PRLs, hence the binding through the Bateman domain was seen to inhibit the in vitro phosphatase activity [23,185].…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…At the same time, Gehring's group proved the appearance of S-phosphorylation on phosphatases of regenerating liver (PRLs), overexpressed in HeLa cells. This modification is located on the catalytic cysteine, and in vivo it is abundant and persistent (Gulerez et al 2016;Zhang et al 2017). Phosphatases of regenerating liver play a causative role in cancer progression and metastasis, and thus deeper insight into the function of S-phosphorylation could be scientifically valuable (Rubio and Köhn 2016;Yu and Zhang 2018).…”