| Description |
The Hedgehog (Hh) pathway is an evolutionarily conserved signaling pathway essential in embryonic development and tissue homeostasis. Several cancers can form as a result of Hh pathway dysfunction. The G protein-coupled receptor (GPCR) Smoothened (SMO) is activated upon binding of the secreted Hh protein to its receptor PTCH1 at the cell surface. SMO activation propagates the signal across the membrane by binding the protein kinase A catalytic subunit (PKA-C) and preventing it from phosphorylating glioma-associated (GLI) transcription factors. Evidence suggests that in vertebrates, PKA-C participates in a protein complex that facilitates GLI phosphorylation and binding to SMO. Whether such a complex exists, as well as the identity of its molecular components, remain unknown. We hypothesize that Kif7, an atypical kinesin, and suppressor of fused (SUFU), a key GLI binding partner and inhibitor, participate in a protein complex with GLI and/or SMO. We tested this hypothesis using bioluminescence resonance energy transfer (BRET), which allows sensitive and specific detection of protein-protein interactions in live mammalian cells. SuFu and Kif7 were found to exhibit BRET with PKA-C. Kif7 was found to exhibit BRET with SMO. Each of these results indicates a protein-protein interaction. This study provides a deeper understanding of how PKA-C binds GLI in the pathway "off "state, and SMO in the pathway "on" state, and takes steps toward delineating the molecular components of a key regulatory protein complex in the Hh pathway. A deeper understanding of PKA-C binding interactions will present a new avenue for treatment of cancers and diseases caused by caused by Hh pathway dysfunction. |
