title: K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin.
abstract: Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.
authors.affiliation: Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 2170, USA. birchena@mail.ncifcrf.gov
authors.forename: Maria C
authors.initials: MC
authors.lastname: Birchenall-Roberts
authors.forename: Tao
authors.initials: T
authors.lastname: Fu
authors.forename: Soo-Gyung
authors.initials: SG
authors.lastname: Kim
authors.forename: Ying K
authors.initials: YK
authors.lastname: Huang
authors.forename: Michael
authors.initials: M
authors.lastname: Dambach
authors.forename: James H
authors.initials: JH
authors.lastname: Resau
authors.forename: Francis W
authors.initials: FW
authors.lastname: Ruscetti
mesh_terms: D002466:Cell Nucleolus / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D019913:Nuclear Localization Signals; D010750:Phosphoproteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000096:biosynthesis* / Q000378:metabolism; D000097796:Nucleolin
chemical_list: D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D016283:Proto-Oncogene Proteins p21(ras); D016601:RNA-Binding Proteins; D018631:ras Proteins; D000097796:Nucleolin; C117307:KRAS protein, human