PRL-3 disrupts epithelial architecture by altering the post-mitotic midbody position.

Disruption of epithelial architecture is a fundamental event during epithelial tumorigenesis. We show that the expression of the cancer-promoting phosphatase PRL-3 (PTP4A3), which is overexpressed in several epithelial cancers, in polarized epithelial MDCK and Caco2 cells leads to invasion and the formation of multiple ectopic, fully polarized lumens in cysts. Both processes disrupt epithelial architecture and are hallmarks of cancer. The pathological relevance of these findings is supported by the knockdown of endogenous PRL-3 in MCF-7 breast cancer cells grown in three-dimensional branched structures, showing the rescue from multiple-lumen- to single-lumen-containing branch ends. Mechanistically, it has been previously shown that ectopic lumens can arise from midbodies that have been mislocalized through the loss of mitotic spindle orientation or through the loss of asymmetric abscission. Here, we show that PRL-3 triggers ectopic lumen formation through midbody mispositioning without altering the spindle orientation or asymmetric abscission, instead, PRL-3 accelerates cytokinesis, suggesting that this process is an alternative new mechanism for ectopic lumen formation in MDCK cysts. The disruption of epithelial architecture by PRL-3 revealed here is a newly recognized mechanism for PRL-3-promoted cancer progression.

The metastasis-promoting phosphatase PRL-3 shows activity toward phosphoinositides.

Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.

Probing Mammalian Cell Size Homeostasis by Channel-Assisted Cell Reshaping.

Cell size homeostasis can be achieved by size checkpoints that couple cell size to cell-cycle progression or by alternative mechanisms such as constant extension. In mammalian cells, the existence of strict size checkpoints remains controversial due to the technical limitations in determining cell size directly and accurately. We developed a microfabricated channel system that linearizes mammalian cell growth and facilitates cell size measurements. By tracking cell length, while directly visualizing cell-cycle progression in rat basophilic leukemia cells and RAW 264.7 macrophages, we examined the mechanisms of size homeostasis and the existence of a size checkpoint at the G1/S transition. Our analysis revealed a two-tier size homeostasis mechanism where a G1 "sizer" or "adder" could operate, depending on the birth size of the cells.

Keep it on the edge: The post-mitotic midbody as a polarity signal unit.

The maintenance of the epithelial architecture during tissue proliferation is achieved by apical positioning of the midbody after cell division. Consequently, midbody mislocalization contributes to epithelial architecture disruption, a fundamental event during epithelial tumorigenesis. Studies in 3D polarized epithelial MDCK or Caco2 cell models, where midbody misplacement leads to multiple ectopic but fully polarized lumen-containing cysts, revealed that this phenotype can be caused by 2 different scenarios: the loss of mitotic spindle orientation or the loss of asymmetric abscission. In addition, we have recently proposed a third cellular mechanism where the midbody mislocalization is achieved through cytokinesis acceleration driven by the cancer-promoting phosphatase of regenerating liver (PRL)-3. Here we critically review these findings, and we furthermore present new data indicating that midbodies themselves might act as signal unit for polarization since they can infer apical characteristics to a basal membrane.

A two-tiered mechanism of EGFR inhibition by RALT/MIG6 via kinase suppression and receptor degradation.

Signaling by epidermal growth factor receptor (EGFR) must be controlled tightly because aberrant EGFR activity may cause cell transformation. Receptor-associated late transducer (RALT) is a feedback inhibitor of EGFR whose genetic ablation in the mouse causes phenotypes due to EGFR-driven excess cell proliferation. RALT inhibits EGFR catalytic activation by docking onto EGFR kinase domain. We report here an additional mechanism of EGFR suppression mediated by RALT, demonstrating that RALT-bound EGF receptors undergo endocytosis and eventual degradation into lysosomes. Moreover, RALT rescues the endocytic deficit of EGFR mutants unable to undergo either endocytosis (Dc214) or degradation (Y1045F) and mediates endocytosis via a domain distinct from that responsible for EGFR catalytic suppression. Consistent with providing a scaffolding function for endocytic proteins, RALT drives EGFR endocytosis by binding to AP-2 and Intersectins. These data suggest a model in which binding of RALT to EGFR integrates suppression of EGFR kinase with receptor endocytosis and degradation, leading to durable repression of EGFR signaling.