Substrate-trapping techniques in the identification of cellular PTP targets

Blanchetot, Christophe; Chagnon, Mélanie J.; Dubé, Nadia; Hallé, Maxime; Tremblay, Michel L.

doi:10.1016/j.ymeth.2004.07.007

Cited by 148 publications

(157 citation statements)

References 72 publications

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“…The assay was performed as previously described, 26,27 with modifications. 293T cells were transfected with Flag vector alone or Flag-tagged PTPRS (WT, C1142S, and D1110A) using Lipofectamine 2000 and cultured for 48 hours.…”

Section: Methodsmentioning

confidence: 99%

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor–induced epithelial‐mesenchymal transition

et al. 2015

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Hepatocellular carcinoma (HCC) is the third-most lethal cancer worldwide. Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients' prognosis. In this study, we report that protein tyrosine phosphatase receptor S (PTPRS) is significantly down-regulated in nearly 80% of HCCs, and its expression negatively correlates with aggressive pathological features, such as larger tumor size and advanced stage. In addition, PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients, although 16.7% of HCCs show intratumor heterogeneous expression of PTPRS. Restoration of wild-type, but not mutant, PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo, whereas knockdown of its expression significantly promotes invasion and metastasis. Notably, PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition (EMT). Moreover, PTPRS forms a complex with epithermal growth factor receptor (EGFR) and regulates its tyrosine residues' phosphorylation. Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS, whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation. Meanwhile, promoter hypermethylation of PTPRS is frequently detected in HCC samples and cell lines. Treatment with a demethylation agent, 5-aza-2 0 -deoxycytidine, recovers PTPRS expression in a dose-dependent manner. Conclusions: Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC. (HEPATOLOGY 2015;62:1201-1214 H epatocellular carcinoma (HCC), epidemic to Asia and Africa with an increasing incidence in Western countries, is the third-leading cause of cancer-related deaths worldwide.1 Less than 30% of HCCs are diagnosed at an early stage and are amenable for resection, liver transplantation, or local ablation.2 In advanced cases, the only effective systemic therapy is the multikinase inhibitor, sorafenib, with a partial response of only 2.2% and median overall survival (OS) of 9.2 months.3 Apart from sorafenib, several novel targeted agents or regimens have been tested; however, none of them showed any positive results, mandating alternative effective treatments.4 Therefore, understanding of the driving events of HCC progression and metastasis is of

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Section: Methodsmentioning

confidence: 99%

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor–induced epithelial‐mesenchymal transition

et al. 2015

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“…67), and a proteomic approach has been used for the identification of substrates of the PTP, PTPN22, by MS analysis of proteins associated with a substrate-trapping mutant (68). In this study, we demonstrate that a substrate-trapping form of Cdc14 can be used to identify and validate novel substrates, including Bni1, Bnr1, and Kar9.…”

Section: Discussionmentioning

confidence: 71%

Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

Bloom

Cristea

Procko

et al. 2011

Journal of Biological Chemistry

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Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.

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“…Wildtype HopZ1a or the catalytic mutant HopZ1a C216A (hereafter HopZ1a C/A ) was also cloned as a fusion to the LexA DNA-binding domain. Catalytically inactive enzymes have been found to exhibit stabilized interactions with substrates (19,20). ZED1 was cloned as a fusion with the B42 activation domain.…”

Section: Ref 1)mentioning

confidence: 99%

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

Lewis

Lee²,

Hassan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

177

245

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Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.ZED1-related kinase | ZRK | hypersensitive response

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Substrate-trapping techniques in the identification of cellular PTP targets

Cited by 148 publications

References 72 publications

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor–induced epithelial‐mesenchymal transition

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor–induced epithelial‐mesenchymal transition

Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

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