Mechanism, function and modulation of oncogenic Cripto signaling.

摘要

Cripto is a multifunctional protein with key roles during embryonic development and tumorigenesis. Cripto's oncogenic functions have been linked to its regulation of TGF-beta ligand signaling via the Smad2/3 pathway and its growth factor activity via the MAPK/PI3K pathways. Notably, Cripto is required for signaling by certain TGF-beta superfamily members such as Nodal, but also antagonizes others such as activin and TGF-beta. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, since these closely related ligands utilize the same receptors. Chapter one addresses this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a non-competitive activin antagonist. The results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, biochemical data reveal complexes containing activin-A, Cripto and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. Furthermore, both Nodal and activin-A share the same binding residues on ActRII. Finally, data presented here demonstrate that the ALK4 ECD has distinct and separable binding sites for activin-A and Cripto. Importantly, ALK4 ECD mutants with disrupted activin-A binding can bind and block Cripto effects on both activin-A and Nodal signaling. We recently demonstrated that Cripto forms a cell surface complex with glucose-regulated protein-78 (GRP78). Chapter two provides evidence that shRNA knockdown or immunoneutralization of GRP78 inhibits the ability of Cripto to function as a Nodal co-receptor, an antagonist of activin and TGF-beta signaling and an activator of MAPK/PI3K pathways. Disrupting the Cripto/GRP78 complex also prevents Cripto from increasing cellular proliferation, downregulating E-Cadherin, decreasing cell adhesion and promoting mitogenic activin-A and Nodal signaling. The results indicate that GRP78 is a critical mediator of Cripto signaling in human tumor, mammary epithelial and embryonic stem cells and suggest that the Cripto/GRP78 complex plays key roles during normal developmental processes and tumorigenesis. Collectively, the work presented in this dissertation represents seminal advances in understanding the mechanism and function of Cripto-mediated signaling.