title: K-Ras4B lipoprotein synthesis: biochemical characterization, functional properties, and dimer formation.
abstract: K-Ras4B, a small GTPase and a key oncogene, plays a central role in the early steps of signal transduction from activated receptor tyrosine kinases by recruiting its downstream effectors to the cell membrane. Specific posttranslational modifications of K-Ras4B, including the addition of C-terminal farnesyl and methyl groups, mediate its proper membrane localization and signaling activity. The mechanism and molecular determinants underlying this selective membrane localization and molecular interactions with its many regulators and downstream effectors are largely unknown. Preparative amounts of the posttranslationally processed K-Ras4B protein are necessary to carry out structural, functional, and cell biological studies of this important oncogene. In this work we describe a simple and efficient method for synthesis of milligram quantities of functionally active, fully processed K-Ras4B. Using this preparation, we observe K-Ras4B dimerization in vitro; this has not been observed previously and could be important for its activity, membrane anchoring, and translocation between different cellular membranes.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 South Ashland Av., Chicago, IL 60607, USA. adement@uic.edu
authors.forename: Alexey
authors.initials: A
authors.lastname: Dementiev
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D054876:Prenylation; D055503:Protein Multimerization; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; C117307:KRAS protein, human; D008074:Lipoproteins; D009842:Oligopeptides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D016283:Proto-Oncogene Proteins p21(ras)