The Conserved CNK CRAC Domain Functions as a Cytohesin-2 Binding Site

Open Access
Schwarz, Margaret C.
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Joseph C. Reese, Thesis Supervisor
  • Lorraine C Santy, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
  • Epithelia
  • migration
  • GTPases
  • cytohesins
Epithelial cells grow as sheets to form barriers that separate different compartments of the body. Though epithelia are usually stationary under normal circumstances, they may become migratory during embryonic development, wound healing, or tumor metastasis. The adoption of a motile phenotype requires regulated changes in the actin cytoskeleton and in the attachment of cells to the substratum. This protein cascade, started by HGF, is tightly regulated. The HGF ligand binds to and activates the c-Met receptor tyrosine kinase, which activates a signaling branch that contains the small GTPase Ras. The pathway downstream of Ras has not been well characterized in literature. CNKs (connector enhancer of KSR) are scaffold proteins that promote Ras to MEK signaling. Mammalian CNKs have a special conserved region, the CRAC domain, in the C-terminal. The function of this region was previously unknown. Pip3-E is homologous to the C-terminal half of mammalian CNKs and was studied extensively in this paper. This research investigates the cytohesin 2 binding location to scaffolding protein Pip3-E/IPCEF. A series of truncation mutants of Pip3-E were created to more precisely localize its cytohesin-binding domain. From the results obtained, it was determined the CRAC domain functions as the cytohesin 2 binding region.