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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
3P01CA054418-09S2
Application #
6218849
Study Section
Project Start
1999-05-01
Project End
2000-04-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
005436803
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Carrano, Andrea C; Dillin, Andrew; Hunter, Tony (2014) A Krüppel-like factor downstream of the E3 ligase WWP-1 mediates dietary-restriction-induced longevity in Caenorhabditis elegans. Nat Commun 5:3772
Altarejos, Judith Y; Montminy, Marc (2011) CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol 12:141-51
Carrano, Andrea C; Liu, Zheng; Dillin, Andrew et al. (2009) A conserved ubiquitination pathway determines longevity in response to diet restriction. Nature 460:396-9
Bres, Vanessa; Yoh, Sunnie M; Jones, Katherine A (2008) The multi-tasking P-TEFb complex. Curr Opin Cell Biol 20:334-40
Fryer, Christy J; White, J Brandon; Jones, Katherine A (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16:509-20
Tutter, A; McAlpine, G S; Jones, K A (1999) Mechanism of chromatin recognition and transcriptional regulation by LEF-1 and the Wnt/Wg-responsive LEF-1:beta-catenin complex. Cold Spring Harb Symp Quant Biol 64:445-52
Bagga, R; Armstrong, J A; Emerson, B M (1998) Role of chromatin structure and distal enhancers in tissue-specific transcriptional regulation in vitro. Cold Spring Harb Symp Quant Biol 63:569-76
Bagga, R; Emerson, B M (1997) An HMG I/Y-containing repressor complex and supercoiled DNA topology are critical for long-range enhancer-dependent transcription in vitro. Genes Dev 11:629-39
Carlsson, P; Waterman, M L; Jones, K A (1993) The hLEF/TCF-1 alpha HMG protein contains a context-dependent transcriptional activation domain that induces the TCR alpha enhancer in T cells. Genes Dev 7:2418-30