title: Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.
abstract: Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland.
authors.forename: Minghui
authors.initials: M
authors.lastname: Li
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kales
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Ke
authors.initials: K
authors.lastname: Ma
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Shoemaker
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Juan
authors.initials: J
authors.lastname: Crespo-Barreto
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Cangelosi
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. panch@ncbi.nlm.nih.gov lipkowis@mail.nih.gov.
authors.forename: Stanley
authors.initials: S
authors.lastname: Lipkowitz
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland. panch@ncbi.nlm.nih.gov lipkowis@mail.nih.gov.
authors.forename: Anna R
authors.initials: AR
authors.lastname: Panchenko
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D020125:Mutation, Missense; D010766:Phosphorylation; D055550:Protein Stability; D050721:Proto-Oncogene Proteins c-cbl / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D013816:Thermodynamics; D014162:Transfection; D054875:Ubiquitination; D002583:Uterine Cervical Neoplasms / Q000201:enzymology* / Q000235:genetics*
chemical_list: D050721:Proto-Oncogene Proteins c-cbl; C512478:EGFR protein, human; D066246:ErbB Receptors; C067419:CBL protein, human