Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain
Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States; Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States
Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States
Carla A Espinoza
Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States
Jennifer M Michaud
Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States
Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, United States
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States; Department of Medicine/Hematology and Oncology, University of California, San Francisco, San Francisco, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, United States
Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, United States
MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.
