title: Homo-oligomerization domains in the lethal(2)giant larvae tumor suppressor protein, p127 of Drosophila.
abstract: The p127 tumor suppressor protein encoded by the lethal(2)giant larvae, l(2)gl, gene of Drosophila melanogaster forms high molecular mass complexes consisting predominantly of p127 molecules. To determine whether p127 can self-assemble in the absence of other binding factors, we analyzed the size of in vitro synthesized p127 by gel filtration and found that p127 is always recovered in a high molecular mass form, demonstrating 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 protein A, which behaves as a monomeric protein, and determined the level of oligomerization of the fused proteins. This study allowed us to map three discrete homo-oligomerization domains, each of approximately 50 amino acid residues in length. These domains, designated as HD-I, HD-II and HD-III, are located between amino acid residues 160 and 204, 247 and 298, and 706 and 749, respectively. Further analysis showed that the 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 proteins 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 structures consistent with the formation of quaternary complexes, whereas protein A fused to a non-self binding domain of p127 appeared monomeric. Our results indicate that p127 alone is able to build quaternary structures 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 proliferation.
authors.affiliation: Department of Developmental Genetics, Deutches Krebsforschungszentrum, Heidelberg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Jakobs
authors.forename: C
authors.initials: C
authors.lastname: de Lorenzo
authors.forename: E
authors.initials: E
authors.lastname: Spiess
authors.forename: D
authors.initials: D
authors.lastname: Strand
authors.forename: B M
authors.initials: BM
authors.lastname: Mechler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D019281:Dimerization; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D007301:Insect Hormones / Q000096:biosynthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins
chemical_list: D029721:Drosophila Proteins; D007301:Insect Hormones; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C081435:l(2)gl protein, Drosophila