1. Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains
- Author
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Jun Ni, Patricia R. Nano, Taylor K. Johnson, Takamasa Kudo, Nancie Mooney, James K. Chen, Janos Demeter, and Peter K. Jackson
- Subjects
Gene isoform ,Science ,Interaction Networks ,Cell Membranes ,Mutant ,Equipment ,Mutagenesis (molecular biology technique) ,Transfection ,Research and Analysis Methods ,Proteomics ,Homology (biology) ,Mice ,Structure-Activity Relationship ,Cell Signaling ,Protein Domains ,Genetics ,Point Mutation ,Animals ,Humans ,Protein Isoforms ,Cilia ,Molecular Biology Techniques ,Protein kinase A ,Molecular Biology ,Transcription factor ,Multidisciplinary ,Chemistry ,Lasers ,GTPase-Activating Proteins ,Biology and Life Sciences ,Membrane Proteins ,Cell Biology ,Cell biology ,Signal Filtering ,Bandpass Filters ,HEK293 Cells ,Optical Equipment ,Membrane protein ,Mutation ,Signal Processing ,Hedgehog Signaling ,NIH 3T3 Cells ,Engineering and Technology ,Medicine ,Cellular Structures and Organelles ,Research Article ,Signal Transduction - Abstract
ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.
- Published
- 2021