The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis

Abstract

The three PRL (phosphatases of regenerating liver) protein tyrosine phosphatases (PRL-1, -2 and -3) have been identified as key contributors to metastasis in several human cancers, yet the molecular basis of their pro-oncogenic property is unclear. Among the subfamily of PRL phosphatases, overexpression of PRL-2 in breast cancer cells has been shown to promote tumor growth by a mechanism that remains to be uncovered. Here we show that PRL-2 regulates intracellular magnesium levels by forming a functional heterodimer with the magnesium transporter CNNM3. We further reveal that CNNM3 is not a phosphorylated substrate of PRL-2, and that the interaction occurs through a loop unique to the CBS pair domains of CNNM3 that exists only in organisms having PRL orthologs. Supporting the role of PRL-2 in cellular magnesium transport is the observation that PRL-2 knockdown results in a substantial decrease of cellular magnesium influx. Furthermore, in PRL-2 knockout mice, serum magnesium levels were significantly elevated as compared with control animals, indicating a pivotal role for PRL-2 in regulating cellular magnesium homeostasis. Although the expression levels of CNNM3 remained unchanged after magnesium depletion of various cancer cell lines, the interaction between endogenous PRL-2 and CNNM3 was markedly increased. Importantly, xenograft tumor assays with CNNM3 and a mutant form that does not associate with PRL-2 confirm that CNNM3 is itself pro-oncogenic, and that the PRL-2/CNNM3 association is important for conferring transforming activities. This finding is further confirmed from data in human breast cancer tissues showing that CNNM3 levels correlate positively with both PRL-2 expression and the tumor proliferative index. In summary, we demonstrate that oncogenic PRL-2 controls tumor growth by modulating intracellular magnesium levels through binding with the CNNM3 magnesium transporter.

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms

“…These results support those in previous studies, which show that disruption of mitochondrial Mg 2+ homeostasis reduces ATP production (46) and cellular Mg 2+ deprivation decreases coupled respiration in cells (47). Notably, we and another group proposed that the PRL regulates energy metabolism by modulating intracellular Mg 2+ through its association with the CNNM Mg 2+ transporter (8,9). Based on this data, PRL2-KO cells appear to store less energy in the form of ATP and dissipate more energy due to defective Mg 2+ flux.…”

“…Beyond previous studies that identified PRL2 functions in cellular Mg 2+ homeostasis (8,9), herein we demonstrate a requirement of PRL2 for sex-dependent and circadian rhythm-dependent energy metabolism ( Figure 9 and Supplemental Figure 7). Since Mg 2+ is a crucial rate-limiting factor for many metabolic effectors, we propose that differential PRL2 expression levels would regulate the concentration of intracellular Mg 2+ to balance cellular energy requirement.…”

“…We previously demonstrated that PRL2 was required for cellular Mg 2+ homeostasis (8). Since changes in Mg 2+ levels affect cellular energy metabolism (1), the metabolic phenotypes of PRL2-KO mice might stem from Mg 2+ alterations.…”

“…Although oncogenic properties of the PRLs leave no doubt, neither biological substrates nor physiological functions have been clearly demonstrated until recently. Notably, previous studies revealed that PRL1, -2, and -3 control intracellular Mg 2+ levels by interacting with the Mg 2+ transporter of the cyclin M (CNNM) family (8,9), and the interaction between PRL2 and CNNM3 is required to promote breast tumor growth through a Mg 2+ -dependent mechanism (10). These findings paved the way to the understanding of PRL functions in a normal physiological context, especially in Mg 2+ -dependent cellular processes.…”

“…pCNNM3-mCherry plasmid was generated by cloning fused human CNNM3 (8) and mCherry cDNA into EcoRV and NotI sites of pcDNA3.1/Zeo(+) (ThermoFisher Scientific). Human PRL2 was cloned in pDONR221 as described previously (8) and shuttled into pDEST53 destination vector (ThermoFisher Scientific) according to the Gateway system protocol to generate the GFP N-terminal-tagged PRL2. HeLa cells were cultured on the 8-well Thermo Scientific Nunc Lab-Tek II Chamber Slide and transfected pGFP-PRL2 and/or pCNNM3-mCherry plasmids using Lipofectamine 2000 (ThermoFisher Scientific).…”