How a cell responds to an extra-cellular signal and mounts an intracellular response plays a pivotal role in the life cycle of every cell. Implicit in this statement is the fact that such signalling mechanisms regulate the growth and differentiation fate of cells. In particular, abherrent signalling cascades or mutations within the proteins comprising such cascades can lead to disease states such as cancer. Much of the information that is conveyed throughout the cell in various signalling cascades propagates in the form of conformational changes in the proteins involved or in changes in the phosphorylation state of signalling proteins. Given that premise, our goal is to provide a stereochemical foundation for the biological roles played by receptor tyrosine phosphatases and other regulators of cell growth, proliferation, and differentiation. Our major tool for conducting these studies is macromolecular x-ray crystallography. We will characterize the structural features of the tandem catalytic domains of receptor tyrosine phosphatase alpha both alone and when complexed with physiologically relevant phosphotyrosine containing substrates. This list includes the inhibitory C-terminal tail of the proto-oncogene cSrc and the C-terminal tail of the second catalytic domain of receptor tyrosine phosphatase alpha involved in the regulation of signalling through the adapter protein Grb2. In addition, we will provide a structural foundation for all receptor tyrosine phosphatases possessing twin catalytic domains by undertaking the crystallization and structural elucidation of the intracellular portion of receptor tyrosine phosphatase alpha possessing two catalytically distinct domains. Given the importance of transcriptional regulation in growth control, we have begun a collaborative effort aimed at determining the three dimensional structure of the transcriptional regulator, IkappaBalpha (human MAD3), both alone and complexed with the p65 domain of NF-kappaB. Given the importance of regulatory phosphorylation of IkappaBalpha we hope that over the long-term we can provide a structural explanation for the effect of phosphorylation on IkappaBalpha. We will employ a two tiered approach in all the proposed work involving a combination of macromolecular x-ray crystallography for high resolution structural analysis and surface plasmon resonance (BIAcore) for the kinetic and thermodynamic analysis of receptor tyrosine phosphatase alpha/phosphopeptide complexes and NF-kappaB/IkappaBalpha complexes. In this regard, we currently have solved the structures of both catalytic domains of receptor tyrosine phosphatase alpha in an uncomplexed state. In terms of the IkappaBalpha project, we now have overexpression and purification systems for IkappaBalpha and NF-kappaB p65 from baculovirus.
