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1.
J Cell Sci ; 109 ( Pt 6): 1359-68, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8799824

ABSTRACT

The p127 tumour suppressor protein encoded by the lethal(2)giant larvae, [l(2)gl], gene of Drosophila melanogaster is a component of a cytoskeletal network distributed in both the cytoplasm and on the inner face of the plasma membrane. The p127 protein forms high molecular mass complexes consisting mainly of homo-oligomerized p127 molecules and at least ten additional proteins. One of these proteins has been recently identified as nonmuscle myosin type II heavy chain. To determine the functional interactions between p127 and other proteins present in the p127 complexes, we analyzed p127 for posttranslational modifications and found that p127 can be phosphorylated at serine residues. In this report we describe the characteristics of a serine kinase which is associated with p127, as judged by its recovery in p127 complexes purified by either gel filtration or immuno-affinity chromatography. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127 and containing five serine residues which are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Insect Hormones/genetics , Insect Hormones/metabolism , Myosin Heavy Chains/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Conserved Sequence , Cytoskeleton/metabolism , Enzyme Activation , Genes, Insect , Genes, Tumor Suppressor , Humans , Insect Hormones/chemistry , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Phosphorylation , Protein Binding , Sequence Homology, Amino Acid
2.
Oncogene ; 11(2): 291-301, 1995 Jul 20.
Article in English | MEDLINE | ID: mdl-7542763

ABSTRACT

Inactivation of the tumour suppressor gene lethal(2) giant larvae (D-lgl) of Drosophila leads to malignant transformation of the presumptive adult optic centers in the larval brain and tumours of the imaginal discs. These malignancies result from the disorganization of a cytoskeletal network in which the D-LGL protein participates. Here we describe the isolation of a cDNA encoding the human homologue to the D-lgl gene designated as hugl. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, we have prepared polyclonal rabbit antibodies against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues.


Subject(s)
Chromosomes, Human, Pair 17 , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila/genetics , Genes, Tumor Suppressor , Myosins/genetics , Proteins , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Blotting, Northern , Chromosome Mapping , Conserved Sequence , Cytoskeletal Proteins/immunology , DNA, Complementary , Gene Expression Regulation , Genes, Insect , Humans , Kidney/physiology , Mice , Molecular Sequence Data , Muscle, Skeletal/physiology , Myosins/chemistry , Placenta/physiology , Protein Serine-Threonine Kinases/metabolism , RNA , Sequence Homology, Amino Acid
3.
J Cell Biol ; 127(5): 1361-73, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7962095

ABSTRACT

Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Our investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins we have analyzed the structure of the cytosolic form of p127. Using gel filtration and immunoaffinity chromatography we found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicated that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, showed that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, we identified one of the proteins by microsequencing as nonmuscle myosin II heavy chain. Further blot overlay assay showed that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/chemistry , Insect Hormones/metabolism , Myosins/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Cell Membrane/chemistry , Cytoskeleton/chemistry , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Genes, Insect , Insect Hormones/chemistry , Insect Hormones/genetics , Insect Hormones/isolation & purification , Molecular Sequence Data , Molecular Weight , Myosins/chemistry , Myosins/isolation & purification , Polymers , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
4.
J Chromatogr ; 577(1): 146-50, 1992 May 20.
Article in English | MEDLINE | ID: mdl-1400733

ABSTRACT

Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.


Subject(s)
Chromatography, High Pressure Liquid/methods , Nucleotides/isolation & purification , Animals , Ions , Rats , Spectrophotometry, Ultraviolet , Tumor Cells, Cultured
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