Multiple domains in ARHGAP36 regulate PKA degradation and Gli activation

ARHGAP36 is a Rho GTPase-activating protein (GAP) family member that contributes to spinal cord development and tumorigenesis. This multidomain protein is composed of N-terminal sequences that vary between splice isoforms, the GAP-like region, and a unique C-terminal domain, and previous studies have revealed an N-terminal arginine-rich region that promotes protein kinase A catalytic subunit (PKA) degradation and Gli transcription factor activation. Although these signaling mechanisms can account for at least some aspects of ARHGAP36-dependent physiology, the functional roles of other structural elements in ARHGAP36 are not well understood. To address this question, we have mapped the ARHGAP36 structure-activity landscape with both domain- and amino-acid-level resolution. We observe that ARHGAP36-mediated Gli activation in cultured mammalian cells can be autoinhibited by isoform-specific N-terminal sequences, which also regulate ARHGAP36 trafficking and the subcellular targeting of PKA pools. ARHGAP36 autoinhibition is counteracted by the GAP-like and C-terminal domains, which promote protein trafficking to the plasma membrane and primary cilium, respectively. In addition, our studies provide evidence that the GAP-like domain conditionally suppresses the N-terminal arginine-rich region and regulates ARHGAP36 binding to several interactors, including the prolyl oligopeptidase-like protein PREPL and the E3 ubiquitin ligase PRAJA2. Thus, multiple domains within ARHGAP36 can modulate its activity state, subcellular localization, and protein binding, potentially providing a means for achieving tissue-specific ARHGAP36 functions.