Phosphorylation/dephosphorylation of PTP-PEST at Serine 39 is crucial for cell migration.

We investigated the molecular details of the role of protein tyrosine phosphatase (PTP)-PEST in cell migration. PTP-PEST knockout mouse embryonic fibroblasts (KO MEFs) and MEF cells expressing a dominant-negative mutant of PTP-PEST showed significant suppression of cell migration compared to MEF cells expressing wild-type PTP-PEST (WT MEFs). Moreover, MEF cells harbouring a constitutively active mutant of PTP-PEST (S39A MEFs) showed a marked decrease in cell migration. In addition, MEF cells with no PTP-PEST or little PTP activity rapidly adhered to fibronectin and made many focal adhesions compared to WT MEF cells. In contrast, S39A MEF cells showed weak adhesion to fibronectin and formed a few focal adhesions. Furthermore, investigating the subcellular localization showed that Ser39-phosphorylated PTP-PEST was favourably situated in the adherent area of the pseudopodia. Therefore, we propose that suppression of PTP-PEST enzyme activity due to Ser39-phosphorylation in pseudopodia and at the leading edge of migrating cells induces rapid and good adherence to the extracellular matrix. Thus, suppression of PTP activity by Ser39-phosphorylation is critical for cell migration. Three amino acid substitutions in human PTP-PEST have been previously reported to alter PTP activity. These amino acid substitutions in mouse PTP-PEST altered the migration of MEF cells in a positive correlation.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells.

Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.