Cytoplasmic retention sites in p190RhoGEF confer anti-apoptotic activity to an EGFP-tagged protein

• Published in: Brain research. Molecular brain research. 2003-09, Vol.117 (1), p.27-38
• Main Authors: Wu, Junhua, Zhai, Jinbin, Lin, Hong, Nie, Zhenying, Ge, Wei-wen, Garcı&#x0301, a-Bermejo, Laura, Muschel, Ruth J., Schlaepfer, William W., Cañete-Soler, Rafaela
• Format: Article
• Language: English
• Subjects: Animals; Anti-apoptosis; Apoptosis - physiology; Binding Sites; Biological and medical sciences; Blotting, Western; Carrier Proteins - metabolism; Cell Aggregation; Cell Death; Cell Line; Cell physiology; Cytoplasm - metabolism; Cytoplasmic retention; DNA-Binding Proteins; Fundamental and applied biological sciences. Psychology; Glutathione Transferase - genetics; Green Fluorescent Proteins; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors - metabolism; In Situ Nick-End Labeling - methods; Intracellular Signaling Peptides and Proteins; Luminescent Proteins - metabolism; Microscopy, Confocal; Miscellaneous; Molecular and cellular biology; Neuronal homeostasis; Nuclear Proteins - metabolism; Nucleocytoplasmic movements; p190RhoGEF; Peptide Fragments - metabolism; Precipitin Tests - methods; Protein Binding; Proto-Oncogene Proteins c-myc - metabolism; Repressor Proteins; Sequence Homology, Amino Acid; Transfection; Two-Hybrid System Techniques
• ISSN: 0169-328X; 1872-6941
• DOI: 10.1016/S0169-328X(03)00263-8

p190RhoGEF is a large multi-functional protein with guanine nucleotide exchange (GEF) activity. The C-terminal region of p190RhoGEF is a highly interactive domain that binds multiple factors, including proteins with anti-apoptotic activities. We now report that transfection of EGFP-tagged p190RhoGEF protects Neuro 2a cells from stress-induced apoptosis and that anti-apoptotic activity is localized to cytoplasmic retention sequences (CRS-1 and CRS-2) in the C-terminal region of p190RhoGEF. Cytoplasmic retention is conferred to an EGFP fluorescent marker when fused to either CRS-1 or CRS-2. Both cytoplasmic retention and anti-apoptotic activity are lost by deleting CRS-1 and CRS-2 in the p190RhoGEF sequence and can be recovered by restoring either CRS-1 or CRS-2 to the EGFP-tagged sequence. Since the CRS-1 and CRS-2 contain the JIP-1 and 14-3-3 binding sites, we propose that anti-apoptotic activity may be conferred by the binding of p190RhoGEF to JIP-1 or 14-3-3, possibly by altering their interactive properties or nucleocytoplasmic movements. Taken together, our findings support a model whereby multiple interactions of p190RhoGEF confer homeostatic properties to differentiated neurons and may link neuronal homeostasis to the regulation of NF-L expression.