The Big Question
Here’s an overview of my project. If you want to learn more, come to hear my chalk talk next week!
Protein-protein interaction between ”Protein X” and ALK-1
Big Picture:
Transforming growth factor beta (TGFβ) is a regulator of multiple cellular processes including proliferation, differentiation, and cell survival. Specific cell surface receptors bind to the TGFβ ligand and initiate an intracellular signaling cascade through the phosphorylation of transcription factors, the Smads. Here is a picture of the canonical TGF-beta signaling pathway. 
(Blobe, GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med. 2000 May 4;342(18):1350-8.)
One specific TGFβ receptor, ALK-1, expressed specifically on endothelial cells is essential for angiogenesis. A mutation of ALK-1 is proposed to result in the disease hereditary hemorrhagic telangiectasia type 2 (HHT-2). HHT is an autosomal dominant disease characterized by vascular dysplasia. ALK-1 has been shown to interact with several intracellular binding partners. One such partner has been identified as the regulatory subunit of “protein X”. Studying how ALK-1 interacts with these other proteins might allow us to better understand the ALK-1 signaling pathway and propose a way to target the pathway and treat such a devastating human disease.
Research Questions:
1) Do “protein X” and ALK-1 interact?
Yes, according to yeast two hybrid screen performed previously and confirmed by GST pull-down and co-immunoprecipitation experiments
2a) Where is the binding site for ALK-1 on “protein X”?
-Hypothesis: somewhere near the C-terminus (similar to other serine/threonine kinases) -What we’re working on now (Week of June 4/11/17): Molecular Cloning of Truncation Mutants of “Protein X”—creating plasmids that contain fragments of “protein X” to identify the location of binding site
PCR –> Restriction digests –> Ligation –> Transformation into bacteria –> Prep DNA –> Confirm our cloning strategy –> Pull-down assay to confirm interaction –> When confirmed, site-directed mutagenesis to further localize
2b) Where is the binding site for ”protein X” on ALK-1?
Conduct site-directed mutagenesis (instead of previous cloning strategy because truncation mutants will be more difficult given the proposed binding site location on ALK-1)
3) What is the significance of binding on ALK-1 signaling and ”protein X” signaling?
- Is there phosphorylation between molecules? (in vitro kinase assay)
-How does “protein X” affect ALK-1 signaling through the Smads?
(Western blot using phospho-specific Smad antibodies/reporter assays)
-Does ALK-1 binding affect “protein X” activity on its targets?
300 targets—several potentials for TGFβ and ”protein X” pathway overlap, such as transcription factor NF-kβ
These may seem like a lot of questions but really the main purpose of all of them is to further investigate the interaction between ALK-1 and “protein X”. Also I don’t think I will be able to work on answering all these questions during the summer program so I will concentrate mostly on Questions 2a/b in finding the location of interaction.
July 30th, 2007 at 8:54 am
Jake
Blog rocks!
August 4th, 2007 at 5:01 am
Harrison
i like it
August 16th, 2007 at 11:21 pm
Mary
nice article, I like it.
August 25th, 2007 at 6:47 am
Sam
very sensible post