MRCKα/β positively regulates Gli protein activity.

Posttranslational modifications (PTMs) are key regulatory events for the majority of signaling pathways. Transcription factors are often phosphorylated on multiple residues, which regulates their trafficking, stability, or transcriptional activity. Gli proteins, transcription factors that respond to the Hedgehog pathway, are regulated by phosphorylation, but the sites and the kinases involved have been only partially described. We identified three novel kinases: MRCKα, MRCKβ, and MAP4K5 which physically interact with Gli proteins and directly phosphorylate Gli2 on multiple sites. We established that MRCKα/β kinases regulate Gli proteins, which impacts the transcriptional output of the Hedgehog pathway. We showed that double knockout of MRCKα/β affects Gli2 ciliary and nuclear localization and reduces Gli2 binding to the Gli1 promoter. Our research fills a critical gap in our understanding of the regulation of Gli proteins by describing their activation mechanisms through phosphorylation.