Charakterisierung eines neuen Proteins, Mapl-1 und seine Rolle in der Regulation der Pax-6 Funktion.

Abstract

Pax proteins comprise a family of highly conserved transcriptional regulators that hold essential roles during embryonic development and organogenesis. The structural motif common to all Pax proteins is the paired box that has been originally identified in the fruitfly Drosophila melanogaster and encodes for a sequence specific DNA binding domain. The murine Pax genes exhibit specific temporal and spatial expression patterns and are involved in different developmental programs in which they occupy high positions in the corresponding regulatory hierarchies. The function of Pax6 has been tightly correlated with the development of the visual system both in vertebrates as well as in invertebrates. Understanding of the function of Pax6 requires elucidation of the molecular mechanisms that underlie its regulation. The present work describes the physical interaction between Pax6 and a novel identified protein Mapl-1. Mapl-1 exhibits a highly overlapping expression pattern to Pax6 especially during the early stages of eye and brain development. Genes similar to Mapl-1 have also been identified on the basis of sequence similarity in the genomes of Drosophila and the nematode Caenorhabditis elegans. Thus Mapl-1 is the founding member of a novel family of evolutionary conserved proteins. Analysis of the interaction between Pax6 and Mapl-1 in vitro identified the paired domain of Pax6 as protein-protein interaction motif. Furthermore binding of Mapl-1 to the Pax6 paired domain prevented its simultaneous interaction with the consensus DNA binding site. This suggests that Mapl-1 might exert an inhibitory effect on the DNA binding properties of the paired domain and therefore on Pax6 function. Analysis of the cellular distribution of Mapl-1 in cultured cells revealed its association with the cellular microtubules while its overexpression induced dramatic changes in microtubule organization leading to the formation of thick bundles. The microtubule association of Mapl-1 is direct and specific since the organization of the other types of cytoskeletal filaments is not affected and leads to increased stability of the microtubule filaments. Hence, Mapl-1 represents a novel "microtubule associated protein", (MAP) since it does not share any substantial sequence similarity to other known MAPs. Mapl-1 can also translocate into the cell nucleus and possesses transcriptional stimulatory potential. The regulation of the cellular localization of Mapl-1 is under the control of nuclear import and export signals. The ability of Mapl-1 to interact both with Pax6 and the microtubules caused in cultured cells the ectopic localization of the otherwise strictly nuclear Pax6 on microtubule filaments. The data presented in this work create for the first time a link between the cytoskeleton and the regulation of Pax protein activity and provide new insights into the molecular mechanisms that control Pax6 function.