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This volume looks at the latest methods used to study and modulate the biological function and mechanisms of SH2 domains. The chapters in this book are organized into five parts. Part One presents methodology aimed at determining the structures and dynamics of SH2 domains and their complexes with phosphopeptides. Part Two discusses techniques to understand and predict interactions of SH2 domains by measuring or calculating their affinity to phosphopeptides. Part Three focuses on inhibitors of SH2 domains that lead the way for chemical tool development and drug discovery. Part Four describes how to evolve and engineer SH2 domains with specific binding properties, and Part Five explores how to measure the regulation of protein tyrosine phosphatase activity through allosteric binding of peptides to SH2 domains and condensation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, SH2 Domains: Functional Modules and Evolving Tools in Biology is a valuable resource for researchers, working in the biophysical and biochemical field, who want to learn more about this exciting and versatile class of regulatory and signaling domains. .
Cytology. --- Biomolecules. --- Physical biochemistry. --- Macromolecules. --- Proteins. --- Cell Biology. --- Structural Biology.
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Dynamic events of protein and lipid phosphorylation and dephosphorylation are involved virtually in all cellular signaling networks and transduction pathways. Kinases are the enzymes that catalyze phosphorylation reactions, while phosphatases are responsible for phosphate hydrolysis from the substrates. Defective or inappropriate activity of phosphatases contributes to the development of many human diseases, including cancer. PRL-3 is a plasma membrane-associated dual specificity phosphatase that exhibits a highly restricted and tightly regulated expression pattern. Additionally, PRL-3 is an emerging prognostic marker for cancer progression and a promising therapeutic target. Although in the last decade numerous clinical studies have extensively validated the causative role of PRL-3 in cancer metastasis, to date its substrate(s) and the direct downstream transduction pathways affected by PRL-3 overexpression are still elusive.The first part of this work concerns the in vitro characterization of PRL-3 phosphatase activity towards phosphopeptides and phosphoinositides. We show that PRL-3 does not present any activity against the library of phosphopeptides tested, while it robustly dephosphorylates the phosphoinositide PtdIns(4,5)P2. Moreover, our experimental results and molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. Finally, structure-activity relationship studies correlate the PRL-3 phosphatase activity toward PI(4,5)P2 with its ability to promote cell migration.The second part of this work concerns the characterization of PRL-3 oncogenic activity with respect to epithelial cell polarity. Using an organotypic 3D-culture system, in which epithelial cells form highly organized spherical cysts, we show that overexpression of PRL-3 significantly affects epithelial morphogenesis leading to the development of cysts with ectopic lumens. Moreover, we prove that a remnant organelle from the cytokinesis process, the post-mitotic midbody, is the first polarization signal during cyst development. Finally, we show that PRL-3 over- expression generates major defects in epithelial cell polarization by interfering with the fate of post-mitotic midbodies.In conclusion, we show that PRL-3 could be a new phosphoinositide phosphatase and that its overexpression affects epithelial cell polarization by altering post-mitotic midbody fate, suggesting a novel mechanism for epithelial tumorigenesis.
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