Structure of a Helical Signaling Domain from p130Cas
EMSL Project ID
2297
Abstract
The cellular responses to integrin signals are coordinated through specific protein-protein interactions. In this project, we focus on an adaptor protein called Cas that becomes phosphorylated upon integrin/ligand binding, forms complexes with a number of signaling proteins and switches on and off downstream signals. The tyrosine-phosphorylation level of Cas correlates well with the transforming phenotype of cells and is thought to play an essential role in the process of cell transformation. Recently, it has been recognized that Cas is identical to the BCAR-1 gene that causes restance to anti-estrogen drugs (tamoxifen) in breast cancer cells. Adaptor proteins such as Cas interact with a number of different molecules. The interacting surfaces between these pairs of proteins are potential targets for drug design, but structural details of the partner molecules must be known. In this study, the first step is to determine the molecular organization of functional regions of the large (130kDa) Cas protein. Cas contains a 'serine-rich' region that is serine-phosphorylated following integrin-mediated cell adhesion. Serine-phosphorylation of the binding partner is important for interactions of this protein with 14-3-3 protein that plays a chaperone role to modulate signal transduction pathways. The Cas/14-3-3 complex may be involved in activation of the Erk pathway. The 3D structure of 14-3-3 is known and the goal of this project is to determine the stucture of Cas serine-rich domain that binds to it. Secondary structure prediction and circular dichroism indicate that the domain is highly helical. Recombinant constructs are in hand to prepare soluble protein for NMR analyses. 1H-15N HSQC and TROSY spectra reveal that the 1H/15N amide resonances are clustered, so the experiments will require data acquisition at high fields employing TROSY to relieve the spectral overlap and improve the resolution. The plan is to record a set of triple resonance experiments using doubly-labeleed protein to obtain assignments and distance restraints for the domain.
Project Details
Project type
Capability Research
Start Date
2001-12-05
End Date
2002-08-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
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