The Perkins Laboratory applies genetic, cellular and molecular tools to fundamental questions in developmental biology. Using Drosophila as an experimental model we continue to contribute insights into the signal transduction pathways essential during embryonic development.
I. Previously, we established that the structurally complex protein tyrosine phosphatase (PTP) Corkscrew (CSW) plays an essential role in the transduction of signals initiated by the activation of receptor tyrosine kinases (RTKs). The importance of CSW function is underscored in csw mutant flies where developmental defects, in tissues whose development requires RTK function, result in lethality. Significantly, CSW has been functionally conserved through evolution since a homologous mammalian PTP, SHP-2, can substitute for CSW during development.
In our efforts to understand the genetic hierarchy and molecular mechanisms of CSW function, we have undertaken several lines of investigation. From our work and others, it is apparent that the precise molecular mechanism of CSW function varies with the RTK; that is, each RTK specific signal differentially uses CSW's various functional domains to achieve transduction. To explore this point, ongoing phenotypic analyses of natural and site-directed csw mutations in vivo are determining which domains of CSW are required for function in specific RTK pathways.
We continue to characterize two proteins that physically interact with CSW. One of these proteins, DIM-7, is a member of the Importin superfamily of known nuclear import proteins. Deletion of the dim-7 gene dramatically reduces nuclear localization of activated MAP kinase. Directly linking DIM-7 to nuclear import, this defect can be rescued by expression of wild type DIM-7. We suggest a model whereby DIM-7 is a member of the import machinery for activated MAP kinase. We further speculate that the DIM-7:CSW association constitutes a regulatory mechanism at the level of the receptor governing MAP kinase nuclear import.
To test this model cell-based cytological, genetic and molecular analyses are elucidating the precise molecular mechanisms by which RTK signaling regulates nuclear import. We have mutated and are analyzing the residues of CSW that are essential to bind DIM-7. In our efforts to understand how DIM-7 recognizes MAP kinase as an import cargo, we have identified, mutated and are analyzing four putative, consensus MAP kinase docking sites in DIM-7. Finally, we have begun to investigate the import specificity of DIM-7 by determining whether or not DIM-7 participates in the nuclear import of another MAP kinase family member, D-JNK.
II. We have initiated a new, multifaceted project involving basic and clinical researchers working to alleviate tragic human malformations. Four integrated projects, involving flies, chicks, rodents, and humans, are designed to reveal molecular components of pulmonary development that when mutated result in pulmonary hypoplasia. Recognizing that the basic mechanisms of cell signaling and tissue morphogenesis are conserved across species, our lab is taking a two-pronged approach.
First, we are genetically identifying and characterizing new components of signaling pathways that operate during development of the fly trachea Previously, using a genetic approach, we undertook a screen that revealed six genomic regions that encode genes whose functions are to enhance CSW, an essential component of tracheal development. We have identified five of these genes and are currently characterizing their effects on developing fly trachea.
Second, we are developing Drosophila as a model genetic system to search for new, or analyze existing pulmonary therapeutics. Using a chemical approach to tracheal growth and differentiation, we have documented effects on tracheal growth and branching with several molecules that have been previously demonstrated to improve growth and differentiation of mouse lungs in organ culture. These results confirm that the fly trachea behaves similarly to these molecules as do developing vertebrate lungs. We are now adapting this assay system to screen collections of small molecules for their abilities to accelerate or suppress tracheal growth.
We anticipate our findings will be applied to vertebrate models of lung hypoplasia, with the hope that our work will be ultimately translated into a viable therapy to alleviate the tragic effects of pulmonary hypoplasia.
Contact Information
Phone: 617-724-1618
Fax: 617-724-2736
