Biochemical Characterization of a Novel KRAS Insertion Mutation from a Human Leukemia*

A novel alteration in exon 1 of KRAS was detected by single strand conformational polymorphism analysis of DNA amplified from the bone marrow of a 4-year-old child with myeloid leukemia. Sequencing of this mutant allele revealed an insertion of three nucleotides between codons 10 and 11 resulting in an in-frame insertion of glycine. Expression of the mutant protein in NIH 3T3 cells caused cellular transformation, and expression in COS cells activated the Ras-mitogen-activated protein kinase signaling pathway. Surprisingly, Ras·GTP levels measured in COS cells established that this novel mutant accumulates to 90% in the GTP state, considerably higher than a residue 12 mutant. Biochemical analysis confirmed that the higher Ras·GTP levels correspond to a dramatic decrease in intrinsic GTP hydrolysis as well as resistance to GTPase-activating proteins. This mutation is the first dominant Ras mutation found in human cancer that does not involve residues 12, 13, or 61, and its biochemical properties should help elucidate the mechanism of oncogenic activation.

[1]  T. Jacks,et al.  Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells , 1996, Nature Genetics.

[2]  T. Pawson,et al.  Vascular system defects and neuronal apoptosis in mice lacking Ras GTPase-activating protein , 1995, Nature.

[3]  S. Aaronson,et al.  A novel insertional mutation in the TC21 gene activates its transforming activity in a human leiomyosarcoma cell line. , 1995, Oncogene.

[4]  F. McCormick,et al.  An essential role for Rac in Ras transformation , 1995, Nature.

[5]  S. Archer,et al.  Preparation of Standards and Measurement of Nitric Oxide, Nitroxyl, and Related Oxidation Products , 1995 .

[6]  K. Shannon,et al.  Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras , 1994 .

[7]  G. Buzard,et al.  Activation of the K-ras gene by insertion mutations in chemically induced rat renal mesenchymal tumors. , 1994, Oncogene.

[8]  Robert A. Weinberg,et al.  Tumour predisposition in mice heterozygous for a targeted mutation in Nf1 , 1994, Nature Genetics.

[9]  N. Copeland,et al.  Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. , 1994, Genes & development.

[10]  C. Denny,et al.  Chronic myelomonocytic leukemia: Tel-a-kinase what Ets all about , 1994, Cell.

[11]  P. O'Connell,et al.  Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. , 1994, The New England journal of medicine.

[12]  M Geyer,et al.  Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras. , 1994, Biochemistry.

[13]  Mark S. Boguski,et al.  Proteins regulating Ras and its relatives , 1993, Nature.

[14]  C. Marshall,et al.  Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function , 1993, Molecular and cellular biology.

[15]  P. Cohen,et al.  Activation of the MAP kinase pathway by the protein kinase raf , 1992, Cell.

[16]  S. Rodenhuis.  ras and human tumors. , 1992, Seminars in cancer biology.

[17]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[18]  M. Wigler,et al.  The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins , 1990, Cell.

[19]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[20]  W. Kabsch,et al.  Three-dimensional structures of H-ras p21 mutants: Molecular basis for their inability to function as signal switch molecules , 1990, Cell.

[21]  J. L. Bos,et al.  ras oncogenes in human cancer: a review. , 1989, Cancer research.

[22]  E. Amann,et al.  Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. , 1988, Gene.

[23]  G. A. Martin,et al.  cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Weinberg,et al.  Activation of Ha-ras p21 by substitution, deletion, and insertion mutations , 1985, Molecular and cellular biology.

[25]  J. Hancock,et al.  Purification of baculovirus-expressed recombinant Ras and Rap proteins. , 1995, Methods in enzymology.

[26]  D. Lowy,et al.  Function and regulation of ras. , 1993, Annual review of biochemistry.

[27]  E. Pai,et al.  The structure of Ras protein: a model for a universal molecular switch. , 1991, Trends in biochemical sciences.