title: BRAF activates and physically interacts with PAK to regulate cell motility.
abstract: Increased p21-activated kinase (PAK) signaling and expression have been identified in the invasive fronts of aggressive papillary thyroid cancers (PTCs), including those with RET/PTC, BRAFV600E, and mutant RAS expression. Functionally, thyroid cancer cell motility in vitro is dependent on group 1 PAKs, particularly PAK1. In this study, we hypothesize that BRAF, a central kinase in PTC tumorigenesis and invasion, regulates thyroid cancer cell motility in part through PAK activation. Using three well-characterized human thyroid cancer cell lines, we demonstrated in all cell lines that BRAF knockdown reduced PAK phosphorylation of direct downstream targets. In contrast, inhibition of MEK activity either pharmacologically or with siRNA did not reduce PAK activity, indicating MEK is dispensable for PAK activity. Inhibition of cell migration through BRAF loss is rescued by overexpression of either constitutive active MEK1 or PAK1, demonstrating that both signaling pathways are involved in BRAF-regulated cell motility. To further characterize BRAF-PAK signaling, immunofluorescence and immunoprecipitation demonstrated that both exogenously overexpressed and endogenous PAK1 and BRAF co-localize and physically interact, and that this interaction was enhanced in mitosis. Finally, we demonstrated that acute induction of BRAFV600E expression in vivo in murine thyroid glands results in increased PAK expression and activity confirming a positive signaling relationship in vivo. In conclusion, we have identified a signaling pathway in thyroid cancer cells which BRAF activates and physically interacts with PAK and regulates cell motility.
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Samantha K
authors.initials: SK
authors.lastname: McCarty
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Saji
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Knippler
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Lawrence S
authors.initials: LS
authors.lastname: Kirschner
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA.
authors.forename: Soledad
authors.initials: S
authors.lastname: Fernandez
authors.affiliation: Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA Division of EndocrinologyDiabetes and Metabolism, Department of Internal MedicineDepartment of Molecular VirologyImmunology, and Medical GeneticsCenter for BiostatisticsThe Ohio State University and Arthur G. James Comprehensive Cancer Center, 565 McCampbell Hall, 1581 Dodd Dr., Columbus, Ohio 43210, USA matthew.ringel@osumc.edu.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Ringel
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051379:Mice; D008938:Mitosis; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D013964:Thyroid Neoplasms / Q000378:metabolism* / Q000473:pathology*; D014407:Tumor Cells, Cultured; D054462:p21-Activated Kinases / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D054462:p21-Activated Kinases
keywords: BRAF; PAK1; cancer biology; cell motility; thyroid cancer