title: Functional impairment of p16(INK4A) due to CDKN2A p.Gly23Asp missense mutation.
abstract: The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16(INK4A) and p14(ARF), involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16(INK4A) properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16(wt), p16(23Asp), or the p16(32Pro) pathogenic variant. We found that p16(23Asp) was less efficient than p16(wt) in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p16(23Asp) in familial melanoma.
authors.affiliation: Section of Oncology, Department of Oncology and Surgical Sciences, University of Padova, via Gattamelata, 64, I-35128 Padova, Italy. mariachiara.scaini@unipd.it
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Scaini
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Rossi
authors.forename: Paula Lobao Antunes
authors.initials: PL
authors.lastname: de Siqueira Torres
authors.forename: Daniela
authors.initials: D
authors.lastname: Zullato
authors.forename: Monia
authors.initials: M
authors.lastname: Callegaro
authors.forename: Cinzia
authors.initials: C
authors.lastname: Casella
authors.forename: Monica
authors.initials: M
authors.lastname: Quaggio
authors.forename: Simona
authors.initials: S
authors.lastname: Agata
authors.forename: Sandro
authors.initials: S
authors.lastname: Malacrida
authors.forename: Vanna
authors.initials: V
authors.lastname: Chiarion-Sileni
authors.forename: Antonella
authors.initials: A
authors.lastname: Vecchiato
authors.forename: Mauro
authors.initials: M
authors.lastname: Alaibac
authors.forename: Marco
authors.initials: M
authors.lastname: Montagna
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Mann
authors.forename: Chiara
authors.initials: C
authors.lastname: Menin
authors.forename: Emma
authors.initials: E
authors.lastname: D'Andrea
mesh_terms: D002453:Cell Cycle; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000502:physiology*; D019942:Genes, p16; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D020125:Mutation, Missense; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D055273:Salivary Proline-Rich Proteins / Q000378:metabolism
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D055273:Salivary Proline-Rich Proteins