Crk-family adaptors mediate the timely formation of protein complexes elicited by a variety of external and intrinsic stimuli and contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks. Despite the fact that adaptor proteins have no intrinsic catalytic or transcriptional activity, their aberrant behavior may lead to oncogenic transformation. Our long-term objectives are to (i) provide the structural basis of Crk-mediated signaling phenomena;(ii) determine the molecular features of the interaction between Crk and Abl, which results in Abl trans-activation;(iii) understand the physicochemical basis of regulatory phenomena in cell signaling;and (iv) design drug candidates that could control aberrant signal transduction events leading to cancer. The strong hypothesis behind the proposed research is that the binding activity of Crk is regulated by a novel autoinhibitory mechanism. Notably, autoinhibition is further controlled by an intrinsic switch afforded by prolyl cis-trans isomerization whose kinetics is effectively catalyzed by cyclophilin A (CypA). We have additionally identified that P221, which lies by the tyrosine residue (Y222) that becomes phosphorylated by c-Abl, undergoes independently cis/trans isomerization that is also catalyzed by CypA.
We aim to: 1. Delineate the structural and dynamic features of both the cis and trans conformers of the linker-SH3C Crk fragment, to understand how cis/trans isomerization at P238 regulates the interaction between the linker and the SH3C domain. 2. Determine the molecular basis of the intramolecular SH3N-SH3C interaction, which forms the basis of an autoinhibitory mechanism. 3. Characterize the structural and dynamic properties of full-length Crk (c-Crkll), to understand how distinct regulatory mechanisms integrate in the context of the physiological cellular form of Crk. 4. Determine the effect of the autoinhibition and proline cis/trans isomerization on the biological function of Crk, both in vitro and in vivo. Crk proteins are involved in mediating many signaling events during the physiological cellular function, but it may also result in tumorigenesis. In this proposal, we will investigate the mechanisms that regulate the activity of the Crk protein, that is, how the protein is switched """"""""on"""""""" and """"""""off."""""""" We hope to ultimately translate the gained knowledge into new strategies for the rational design of drug candidates that could inhibit proliferation of cancerous cells.
