UCSF

Cells communicate with their environment through the transmission of external stimuli internally by a complex, interconnected network of signaling pathways. The magnitude, duration, and location of these pathways vary throughout the stages of development and adulthood to give rise to discrete features and to maintain homeostasis at the cellular, tissue, and organismal level. Through a countless number of studies, our knowledge of cellular signaling pathways has grown immensely in the past few decades. However, an area of study that remains largely understudied is the roles cellular regulators play in fine-tuning the output and dynamics of these pathways. Mounting evidence from studies on malignancies such as cancer, which take advantage of and hijack cellular signaling pathways, have illustrated the need to characterize such regulatory factors in addition to the pathway driving factors that have been the target of much research to date. Increasing difficulties in pharmaceutical development highlight the need to expand the range of druggable targets, and recent studies suggest that these proteins are well posed to become the next generation of drug targets. Here, we present the functional characterization of two understudied regulatory proteins: Canopy 4 (CNPY4) and PEAK3. We show that CNPY4 is a member of the saposin-like protein (SAPLIP) family that intersects the Sonic hedgehog (SHH) signaling pathway. Silencing of Cnpy4 leads to severe hyperactivation of the SHH pathway and alteration of accessible cholesterol levels in the cell membrane. These changes likely contribute to the severe congenital malformations observed in Cnpy4 knockout mice. We further demonstrate that PEAK3, a novel pseudokinase with no previously reported function, modulates cellular motility and migration through antagonization of the adaptor protein CrkII. PEAK3 regulation of CrkII is dependent on its ability to dimerize through a structural motif termed the split helical dimerization domain and the presence of an intact DFG motif. Furthermore, our initial studies suggest that PEAK3 antagonization of CrkII may itself be controlled by its interaction with the 14-3-3 family of regulatory proteins. Together, our studies provide the first functional evidence that CNPY4 is a regulator of the SHH pathway and that PEAK3 is a regulator of the CrkII pathway.