Dbl-Like Regulators of Small G-Proteins

Zheng, Yi

Abstract

The long-term objectives of this project are to understand the structure- function relation of Dbl oncoprotein and to determine the mechanisms of signal transduction directly involving Dbl and Dbl-like molecules. The dbl oncogene product is a cytoskeletal-associated protein functioning as guanine nucleotide exchange factor (GEF) for Rho-type small GTP-binding proteins. Oncogenic activation of Dbl oncoprotein occurs as a result of amino-terminal truncation of a vimentin-like domain from the 115 kDa proto- dbl product. A region of over 200 amino acids in Dbl, designated as Dbl- homology (DH) domain, is directly responsible for the GEF activity; and the DH domain together with its immediate downstream pleckstrin homology (PH) domain constitutes the minimum unit required for transforming function. This structural arrangement of DH domain in tandem with a PH domain has been found in a growing family of growth regulatory molecules for which Dbl represents a prototype. For the most part, little is known regarding the mechanisms by which Dbl and related proteins elicit their functions in the regulations of cell growth and transformation. To dissect the structural elements in proto- and oncogenic Dbl proteins responsible for the transforming function and to begin to delineate the immediate downstream signaling mechanism, three areas of studies will be pursued in this proposal with each area comprising a specific aim.
The first aim i nvolves the examination of the structural features of the Dbl-homology domain in Dbl and related proteins in the regulation of the activities of Rho-type GTP-binding proteins. We will determine the structural elements of the DH domain directly involved in G-protein binding and/or catalysis and generate dominant negative forms of Dbl and related proteins. This data will correlate with the studies on signaling mechanism of these molecules in which we will determine whether Dbl and related Cdc24 protein would transmit their transformation/growth signals through specific members of Rho-type GTPases.
The second aim will address the role of the pleckstrin- homology domain of Dbl and Dbl-like regulatory molecules in their cellular functions. We will examine the specificity of the PH domain function by mutagenic and chimeric approaches and determine its pattern of cellular localization by microinjection. In the third specific aim we will elucidate the mechanism of regulation for proto-Dbl. One line of approach to understand the drastically reduced transforming capability of proto-Dbl will be to examine the role of the amino-terminal vimentin-like constraining domain of proto-Dbl and compare with the corresponding regions of other members of Dbl-family proteins such as proto-Vav. We will identify the inhibitory mechanism for proto-Dbl and search for a proto-Dbl inhibitor. These detailed characterizations of the Dbl and proto-Dbl proteins represent an essential step toward understanding the mechanism of functions for the family of Dbl-like regulators of the Rho-type small GTP- binding proteins.