ABSTRACT
Guanine nucleotide exchange factors (GEFs) activate the Rho GTPases by accelerating their GDP/GTP exchange rate. Some RhoGEFs have been isolated based on their oncogenic potency, and strategies to inhibit their activity are therefore actively being sought. In this study we devise a peptide inhibitor screening strategy to target the GEF activity of Tgat, an oncogenic isoform of the RhoGEF Trio, based on random mutations of the Trio inhibitor TRIP alpha, which we previously isolated using a peptide aptamer screen. This identifies one peptide, TRIP(E32G), which specifically inhibits Tgat GEF activity in vitro and significantly reduces Tgat-induced RhoA activation and foci formation. Furthermore, subcutaneous injection of cells expressing Tgat and TRIP(E32G) into nude mice reduces the formation of Tgat-induced tumors. Our approach thus demonstrates that peptide aptamers are potent inhibitors that can be used to interfere with RhoGEF functions in vivo.
Subject(s)
Aptamers, Peptide/metabolism , Aptamers, Peptide/pharmacology , Cell Transformation, Neoplastic/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Aptamers, Peptide/chemistry , Female , Guanine Nucleotide Exchange Factors/genetics , Humans , Mice , Mice, Inbred BALB C , NIH 3T3 Cells , Peptide Library , Point Mutation , Protein Serine-Threonine Kinases/genetics , Protein Structure, TertiaryABSTRACT
The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.
Subject(s)
Growth Cones/physiology , Guanine Nucleotide Exchange Factors/physiology , Nerve Growth Factors/physiology , Neuropeptides/physiology , Phosphoproteins/physiology , Protein Serine-Threonine Kinases/physiology , Receptors, Cell Surface/physiology , Tumor Suppressor Proteins/physiology , rac GTP-Binding Proteins/physiology , Adaptor Proteins, Signal Transducing , Animals , Brain/abnormalities , Brain/cytology , Brain/embryology , COS Cells , Cell Line , Cells, Cultured/cytology , Chlorocebus aethiops , DCC Receptor , Enzyme Activation , Female , Growth Cones/ultrastructure , Guanine Nucleotide Exchange Factors/deficiency , Guanine Nucleotide Exchange Factors/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Netrin-1 , Oncogene Proteins/physiology , Phosphoproteins/deficiency , Phosphoproteins/genetics , Protein Interaction Mapping , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Spinal Cord/cytology , Spinal Cord/embryology , p21-Activated Kinases/physiology , rac1 GTP-Binding ProteinABSTRACT
Geminin is an important cell cycle regulator having a dual role in cell proliferation and differentiation. During proliferation, Geminin controls DNA synthesis by interacting with the licensing factor Cdt1 and interferes with the onset of differentiation by inhibiting the activity of transcription factors such as Hox and Six3. During early development Geminin also functions as neural inducer. Thus differential interaction of Geminin with Cdt1 or development-specific transcription factors influence the balance between proliferation and differentiation. Here, we report an additional feature of Geminin showing that it is a novel substrate of caspase-3 during apoptosis in in vitro Xenopus egg extracts. We also show that cleavage of Geminin occurs both in solution and on chromatin with distinct kinetics. In addition we show that cleavage of Geminin by caspase-3 is not relevant to its function as regulator of DNA synthesis, suggesting that its cleavage may be relevant to its role in differentiation.
