THEMIS is a substrate and allosteric activator of SHP1, playing dual roles during T cell development.

THEMIS plays an indispensable role in T cells, but its mechanism of action has remained highly controversial. Using the systematic proximity labeling methodology PEPSI, we identify THEMIS as an uncharacterized substrate for the phosphatase SHP1. Saturated mutagenesis assays and mass spectrometry analysis reveal that phosphorylation of THEMIS at the evolutionally conserved Tyr34 residue is oppositely regulated by SHP1 and the kinase LCK. Similar to THEMIS-/- mice, THEMISY34F/Y34F knock-in mice show a significant decrease in CD4 thymocytes and mature CD4 T cells, but display normal thymic development and peripheral homeostasis of CD8 T cells. Mechanistically, the Tyr34 motif in THEMIS, when phosphorylated upon T cell antigen receptor activation, appears to act as an allosteric regulator, binding and stabilizing SHP1 in its active conformation, thus ensuring appropriate negative regulation of T cell antigen receptor signaling. However, cytokine signaling in CD8 T cells fails to elicit THEMIS Tyr34 phosphorylation, indicating both Tyr34 phosphorylation-dependent and phosphorylation-independent roles of THEMIS in controlling T cell maturation and expansion.

Allosterically inhibited PFKL via prostaglandin E2 withholds glucose metabolism and ovarian cancer invasiveness.

Metastasis is the leading cause of high ovarian-cancer-related mortality worldwide. Three major processes constitute the whole metastatic cascade: invasion, intravasation, and extravasation. Tumor cells often reprogram their metabolism to gain advantages in proliferation and survival. However, whether and how those metabolic alterations contribute to the invasiveness of tumor cells has yet to be fully understood. Here we performed a genome-wide CRISPR-Cas9 screening to identify genes participating in tumor cell dissemination and revealed that PTGES3 acts as an invasion suppressor in ovarian cancer. Mechanistically, PTGES3 binds to phosphofructokinase, liver type (PFKL) and generates a local source of prostaglandin E2 (PGE2) to allosterically inhibit the enzymatic activity of PFKL. Repressed PFKL leads to downgraded glycolysis and the subsequent TCA cycle for glucose metabolism. However, ovarian cancer suppresses the expression of PTGES3 and disrupts the PTGES3-PGE2-PFKL inhibitory axis, leading to hyperactivation of glucose oxidation, eventually facilitating ovarian cancer cell motility and invasiveness.

FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells.

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85ß, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

CRISPR system: Discovery, development and off-target detection.

As a revolutionary gene editing tool based on the adaptive immune defense mechanism of bacteria and archaea against exogenous DNA invasion, CRISPR/Cas system shows many remarkable characteristics over ZFNs and TALENs. However, off-target effect remains as one of the major imperfection of CRISPR/Cas system, hindering its further application in translational research. In this review, we highlight major breakthrough cross the development/application of this powerful toolkit, and summarize feasible methods for detecting potential off-target effects during genetic manipulation. We hope this review will assist scientists for accurate genomic editing in their future research.

Protein tyrosine phosphatase receptor type R (PTPRR) antagonizes the Wnt signaling pathway in ovarian cancer by dephosphorylating and inactivating ß-catenin.

Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/ß-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/ß-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of ß-catenin on Tyr-142, a key site controlling the transcriptional activity of ß-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, ß-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating ß-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.