A research program will be undertaken in the area of TGF-p signaling, a fundamental cellular process that is implicated in multiple human diseases, including cancer. A key feature of the signaling pathway is phosphorylation of a family of latent transcription factors termed R-Smads, a biochemical event that leads to nuclear accumulation. The central hypothesis of this research is that continuous nucleocytoplasmic shuttling of R-Smads with repeated cycles of receptor-mediated phosphorylation and nuclear dephosphorylation, permits constant sensing of the activation status of the receptor and hence efficient termination of signaling upon receptor inactivation. To explore this, we will study the three critical phases in the biochemical lifetime of the R-Smad, Smad2, namely, bis-phosphorylation of the protein by the activated TpR-l receptor, nuclear import of the phosphorylated Smad2/Smad4 complex, and nuclear dephosphorylation of Smad2 followed by its nuclear export. Key to this research program is our ability to introduce biochemical and biophysical probes site-specifically into phosphorylated forms of T[3R-I and Smad2, thereby allowing us to control and monitor their activities. Chemistry-driven protein engineering approaches will be used in conjunction with established biophysical and cell biological approaches to study the detailed mechanisms by which Smad2 interacts with the activated receptor complex, and thereafter shuttles to and from the nucleus.
The specific aims are: 1. To Study the Mechanisms Underlying R-Smad Activation: We will use biochemical and structural techniques to investigate how activated TpR-l receptor recognizes and then double phosphorylates Smad2. 2. To Study the Mechanisms Underlying R-Smad Nuclear Import: We will use biochemical and cell biological techniques to study the molecular mechanisms by which phosphorylation of Smad2 leads to its nuclear accumulation. 3. To Study the Mechanisms Underlying R-Smad Nuclear Export. We will identify and characterize the putative nuclear phosphatase responsible for removing the activation phosphates from Smad2. By providing a more complete quantitative and structural understanding of the basic signaling pathway, this research program will lay a firmer foundation for the rational design of small molecular therapies based on manipulation of TGF-psignaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055843-12
Application #
7390225
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Flicker, Paula F
Project Start
1997-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
12
Fiscal Year
2008
Total Cost
$357,736
Indirect Cost
Name
Rockefeller University
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Cho, Jae-Hyun; Muralidharan, Vasant; Vila-Perello, Miquel et al. (2011) Tuning protein autoinhibition by domain destabilization. Nat Struct Mol Biol 18:550-5
Vila-Perello, Miquel; Muir, Tom W (2010) Biological applications of protein splicing. Cell 143:191-200
Frutos, Silvia; Goger, Michael; Giovani, Baldissera et al. (2010) Branched intermediate formation stimulates peptide bond cleavage in protein splicing. Nat Chem Biol 6:527-33
Chiang, Kyle P; Jensen, Mette S; McGinty, Robert K et al. (2009) A semisynthetic strategy to generate phosphorylated and acetylated histone H2B. Chembiochem 10:2182-7
Lockless, Steve W; Muir, Tom W (2009) Traceless protein splicing utilizing evolved split inteins. Proc Natl Acad Sci U S A 106:10999-1004
Ceccarini, Giovanni; Flavell, Robert R; Butelman, Eduardo R et al. (2009) PET imaging of leptin biodistribution and metabolism in rodents and primates. Cell Metab 10:148-59
Valiyaveetil, Francis I; Sekedat, Matthew; MacKinnon, Roderick et al. (2006) Structural and functional consequences of an amide-to-ester substitution in the selectivity filter of a potassium channel. J Am Chem Soc 128:11591-9