An Analog-Sensitive Tau Tubulin Kinase 2 Enables Acute Chemical-Genetic Control of Primary Cilia

Abstract Kinases regulate a broad spectrum of cellular processes, including the formation of primary cilia, microtubule-based organelles that protrude from the cell surface and play important roles in development and disease. TTBK2 is a key ciliary kinase that controls primary cilium assembly and maintenance. However, highly selective small-molecule inhibitors of TTBK2 are still lacking, representing a major bottleneck for investigating the temporal requirements and dynamics of TTBK2-dependent processes in cilia. Here, we report the first analog-sensitive variant of TTBK2, enabling rapid, reversible, and highly specific control of TTBK2 kinase activity in ciliogenesis-relevant hTERT-RPE1 cells. Using this system, we define the kinetics of CEP164 phosphorylation, cilium shortening and reassembly, and IFT88 retention at the ciliary base following acute TTBK2 inhibition and subsequent inhibitor washout. Together, our findings provide new insight into TTBK2-dependent pathways and establish analog-sensitive TTBK2 as a powerful tool for investigating the dynamics of TTBK2-regulated processes with high temporal resolution.