HOMOOLIGOMERIZATION DOMAINS IN THE LETHAL(2)GIANT LARVAE TUMOR-SUPPRESSOR PROTEIN, P127 OF DROSOPHILA

The p127 tumor suppressor protein encoded by the lethal(2)giant larvae , l(2)gl, gene of Drosophila melanogaster forms high molecular mass co mplexes consisting predominantly of p127 molecules. To determine wheth er p127 can self-assemble in the absence of other binding factors, we analyzed the size of in vitro synthesized p127 by gel filtration and f ound that p127 is always recovered in a high molecular mass form, demo nstrating that p127 can oligomerize on its own. Previous studies have revealed that p127 may contain three homo-oligomerization domains. To more accurately delineate these domains, we have generated a series of 32 chimaeric proteins made of defined portions of p127 fused to prote in A, which behaves as a monomeric protein, and determined the level o f oligomerization of the fused proteins. This study allowed us to map three discrete homo-oligomerization domains, each of similar to 50 ami no acid residues in length. These domains, designated as HD-I, IID-II and HD-III, are located between amino acid residues 160 and 204, 247 a nd 298, and 706 and 749, respectively. Further analysis showed that th e HD-I and HD-II domains can bind to themselves and to each other. We also mapped a domain in p127 between amino acid residues 377 and 438, which strongly reduces the degree of multimerization of chimaeric prot eins containing HD-I and/or HD-II. Electron microscopy examination of negatively stained chimaeric proteins showed that protein A fused with either the domain HD-II or the domain HD-III forms discrete structure s consistent with the formation of quaternary complexes, whereas prote in A fused to a non-self binding domain of p127 appeared monomeric. Ou r results indicate that p127 alone is able to build quaternary structu res forming a network with which other proteins associate. As revealed by the tumorous phenotype resulting from the inactivation of the l(2) gl gene, the organization of the p127 network and its association with other proteins play critical roles in the control of cell proliferati on. (C) 1996 Academic Press Limited