PROJECT SUMMARY Hedgehog (Hh) signaling is widely involved in development and cancers, including medulloblastoma (MB), the malignant pediatric brain tumor. Current treatment regimens bring life-long devastating side effects to survivors, generating a huge burden to the patients’ family. The current Hh pathway inhibitors are challenged by drug resistance and tumor relapse. We aim to understand the basic transduction mechanisms of the Hh pathway in order to provide new clues to treat Hh-related cancers. This proposal is based on my previous discovery that PDE4D controls local PKA activity at specific subcellular sites to regulate Hh signaling. In the past two years of this grant, we have studied the control of Hh signaling by local PDE4D-PKA at the centrosome. We found that 1) PDE4D3 is anchored to the centrosome by myomegalin to locally inhibit PKA activity, 2) knocking out myomegalin with shRNA or CRISPR/Cas9 specifically increases PKA activity at the centrosome, and 3) this selectively inhibits Hh signaling without interfering other PKA-related cellular events. Based on the exciting results and the innovative tools we have built, in the current proposal we will expand our research to systemically investigate the regulation of Hh signaling by local PKA in the cilium. We will 1) define the signaling mechanism from Smo to PKA in the cilium, a mysterious step in the Hh pathway; 2) determine the effects of selectively promoting ciliary PKA on Hh signaling and Hh-related tumor growth; and 3) identify the signaling transducers associated with Smo via proximity labeling-based proteomics to thoroughly illuminate its downstream signaling cascade during Hh transduction. Our approach is innovative because it combines cutting edge techniques to elucidate the long-standing questions in the Hh pathway. Its significance is underlined by the expectation that it will highlight new approaches to treat pediatric brain tumor with fewer side effects. Equally importantly, this research funding will allow me to engage first generation college students in biomedical research at UC Merced to diversify and strengthen academic research in the economically deprived California Central valley.