Identification of sequences responsible for lost of activity of the human Tre2 oncogene product by functional complementation of the yeast msb3 msb4 mutant.
Christelle Bizimungu, Daniel Portetelle, Micheline Vandenbol
Unite de Biol Anim et Microb, FUSAGx, Ave M. Juin, 6, Gembloux, 5030, Belgium
The yeast proteins Msb3p/Gyp3p and Msb4p/Gyp4p are two Ypt/Rab-specific GTPase-activating proteins (GAPs) involved in exocytosis and organization of the actin cytoskeleton. Msb3p and Msb4p are similar to the product of the human Tre2 oncogene, implicated in a wide variety of human cancer including Ewing's sarcoma, and to the human protein RN-tre which is both the Rab5-GAP in the EGF receptor endocytosis and a Rab5 effector in an actin remodeling pathway. We showed in a previous work (Bizimungu et al., 2003) that the oncogene, unlike RN-tre, is inactive when expressed in an msb3 msb4 yeast mutant. Tre2 (786 aa) and RN-tre (828 aa) share a high degree of similarity on their 460 first amino acids, including the TBC domain (active domain of Ypt/RabGAPs). In order to identify the regions of Tre2 responsible for lost of activity, chimerical proteins were created by sequence exchanges between RN-tre and Tre2, and were tested for their ability to complement the yeast msb3 msb4 double mutation for all the phenotypes observed (perturbed budding pattern, delocalization of actin cytoskeleton, sensitivity of growth to caffeine and DMSO). In conclusion, these experiments localized two regions of the Tre2 sequence involved in lack of activity.