Cdc42 and Ras cooperate to mediate cellular transformation by intersectin-L.
Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.
Wang
Jian-Bin
JB
Department of Molecular Medicine, Veterinary Medical Center, Cornell University, Ithaca, New York 14853, USA.
Wu
Wen Jin
WJ
Cerione
Richard A
RA
D000199:Actins / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000362:Agar / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D051379:Mice; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D000199:Actins; D033942:Adaptor Proteins, Vesicular Transport; D004791:Enzyme Inhibitors; C115919:intersectin 1; D006153:Guanosine Diphosphate; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D000362:Agar; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D020764:cdc42 GTP-Binding Protein; D018631:ras Proteins