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1.
Biochem J ; 352 Pt 2: 425-33, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11085936

ABSTRACT

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.


Subject(s)
Integrin beta1/metabolism , Isoenzymes/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Kinase C/metabolism , Tumor Suppressor Proteins , Cell Line , Enzyme Activation , Humans , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Kinase C-delta
2.
Clin Cancer Res ; 6(8): 3271-81, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10955813

ABSTRACT

H-Ryk is an atypical receptor tyrosine kinase that is expressed in a differentiation-specific manner in epithelial tissues. We have previously shown by in situ hybridization and immunohistochemistry that H-Ryk is overexpressed in malignant ovarian tumors. In addition, we have demonstrated that overexpression of H-Ryk is transforming in vitro and in vivo. To evaluate whether expression of H-Ryk is a prognostic factor in epithelial ovarian cancer, we carried out a retrospective study of 88 primary malignant ovarian tumors (28 serous tumors, 11 mucinous tumors, 29 endometrioid tumors, 13 clear cell tumors, 3 malignant mixed Mullerian tumors, 1 mixed epithelial tumor, 1 primary peritoneal tumor, 1 undifferentiated tumor, and 1 transitional carcinoma) diagnosed between 1990 and 1993 using immunohistochemistry. On univariate analysis, overall survival decreased significantly with age (P = 0.01); in patients with International Federation of Gynecology and Obstetrics (FIGO) stage II (P = 0.008), FIGO stage III (P < 0.001), and FIGO stage IV (P < 0.001) disease; and in patients with residual disease (residual disease < or = 2 cm, P = 0.007; residual disease > 2 cm, P < 0.001) after surgery. In addition, overexpression of the H-Ryk receptor in malignant epithelium (P = 0.04) and blood vessel (P = 0.01) was associated with a significantly decreased overall survival. H-Ryk blood vessel overexpression (P = 0.03), residual disease > 2 cm (P = 0.006), and residual disease < or = 2 cm (P = 0.01) conferred a significantly shorter progression-free survival. No correlation was found between H-Ryk overexpression and age, histological subtype, degree of differentiation, FIGO stage, or residual disease. Overall, after adjustment for all of the prognostic factors by multivariate analysis (Cox proportional hazards model), residual disease was the most powerful prognostic indicator for overall survival (P < 0.001) and progression-free survival (P = 0.01) in this patient subset. This implies that H-Ryk acts cooperatively with other biological factors in the pathogenesis of ovarian cancer.


Subject(s)
Ovarian Neoplasms/enzymology , Receptor Protein-Tyrosine Kinases/biosynthesis , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Endothelium, Vascular/enzymology , Epithelium/enzymology , Female , Humans , Middle Aged , Multivariate Analysis , Muscle, Smooth, Vascular/enzymology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/pathology , Ovarian Neoplasms/surgery , Receptor Protein-Tyrosine Kinases/genetics , Retrospective Studies , Stromal Cells/enzymology , Survival Analysis
3.
Mol Cell Biol ; 19(9): 6427-40, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10454588

ABSTRACT

H-Ryk is an atypical receptor tyrosine kinase which differs from other members of this family at a number of conserved residues in the activation and nucleotide binding domains. Using a chimeric receptor approach, we demonstrate that H-Ryk has impaired catalytic activity. Despite the receptor's inability to undergo autophosphorylation or phosphorylate substrates, we demonstrate that ligand stimulation of the chimeric receptor results in activation of the mitogen-activated protein kinase pathway. The ability to transduce signals is abolished by mutation of the invariant lysine (K334A) in subdomain II of H-Ryk. Further, by in vitro mutagenesis, we show that the amino acid substitutions in the activation domain of H-Ryk account for the loss of catalytic activity. In addition to the essential aspartate residue, either phenylalanine or glycine is required in the activation domain to maintain proper conformation of the catalytic domain and thus ensure receptor autophosphorylation. Homology modelling of the catalytic domain of H-Ryk provides a rationale for these findings. Thus, the signalling properties of H-Ryk are divergent from those of other classical receptor tyrosine kinases.


Subject(s)
Receptor Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Catalytic Domain/genetics , Cell Line, Transformed , DNA Primers/genetics , Enzyme Activation , Humans , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Phosphorylation , Point Mutation , Protein Conformation , Receptor Protein-Tyrosine Kinases/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction
4.
Cancer Res ; 59(10): 2265-70, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10344726

ABSTRACT

Abnormalities in the function of receptor tyrosine kinases (RTKs) have been demonstrated to be important in the pathogenesis of cancer. H-Ryk, a new member of the RTK family, is an unusual RTK in that it is catalytically inactive because of amino acid substitutions of conserved residues in the catalytic domain. We show by immunohistochemistry that it is expressed in the epithelium, stroma, and blood vessels of normal tissues. Evaluation of a panel of 33 primary ovarian tumors (2 benign, 8 borderline, and 23 malignant) was performed. H-Ryk was overexpressed in borderline and malignant ovarian tumors. In serous and clear cell subtypes, there was increased expression in the epithelium, stroma, and blood vessels. Consistent with this observation, overexpression of H-Ryk in the mouse fibroblast cell line NIH3T3 induces anchorage-independent growth and tumorigenicity in nude mice. This implies that overexpression of the receptor can be transforming and may therefore be significant in the pathogenesis of ovarian cancer.


Subject(s)
3T3 Cells/enzymology , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Ovarian Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , 3T3 Cells/transplantation , Adenocarcinoma, Clear Cell/enzymology , Adenocarcinoma, Clear Cell/genetics , Adenocarcinoma, Clear Cell/pathology , Adenocarcinoma, Mucinous/enzymology , Adenocarcinoma, Mucinous/genetics , Adenocarcinoma, Mucinous/pathology , Animals , Blood Vessels/enzymology , Carcinoma, Endometrioid/enzymology , Carcinoma, Endometrioid/genetics , Carcinoma, Endometrioid/pathology , Cystadenocarcinoma, Serous/enzymology , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Cystadenoma, Mucinous/enzymology , Cystadenoma, Mucinous/genetics , Cystadenoma, Mucinous/pathology , Cystadenoma, Serous/enzymology , Cystadenoma, Serous/genetics , Cystadenoma, Serous/pathology , Enzyme Induction , Epithelial Cells/enzymology , Female , Mice , Mice, Nude , Neoplasm Proteins/genetics , Neoplasm Transplantation , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/genetics , Stromal Cells/enzymology , Transfection , Tumor Cells, Cultured
5.
Cancer Metastasis Rev ; 16(1-2): 81-107, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9156281

ABSTRACT

The recent advances in the understanding of the pathogenesis of ovarian cancer have been helpful in addressing issues in diagnosis, prognosis and management. The study of ovarian tumours by novel techniques such as immunohistochemistry, fluorescent in situ hybridisation, comparative genomic hybridisation, polymerase chain reaction and new tumour markers have aided the evaluation and application of new concepts into clinical practice. The correlation of novel surrogate tumour specific features with response to treatment and outcome in patients has defined prognostic factors which may allow the future design of tailored therapy based on a molecular profile of the tumour. These have also been used to design new approaches to therapy such as antibody targeting and gene therapy. The delineation of roles of c-erbB2, c-fms and other novel receptor kinases in the pathogenesis of ovarian cancer has led initially to the development of anti-c-erbB2 monoclonal antibody therapy. The discovery of BRCA1 and BRCA2 genes will have an impact in the diagnosis and the prevention of familial ovarian cancer. The important role played by recessive genes such as p53 in cancer has raised the possibility of restoration of gene function by gene therapy. Although the pathological diagnosis of ovarian cancer is still confirmed principally on morphological features, addition of newer investigations will increasingly be useful in addressing difficult diagnostic problems. The increasingly rapid pace of discovery of genes important in disease, makes it imperative that the evaluation of their contribution in the pathogenesis of ovarian cancer is undertaken swiftly, thus improving the overall management of patients and their outcome.


Subject(s)
Genes, Tumor Suppressor , Genes, erbB , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Proto-Oncogenes , Biomarkers, Tumor/analysis , Female , Genes, Dominant , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Neoplasm Metastasis , Neovascularization, Pathologic , Ovarian Neoplasms/therapy , Polymerase Chain Reaction , Prognosis
6.
Genome Res ; 6(7): 620-7, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8796349

ABSTRACT

The discoidin domain receptor (DDR) is a new class of receptor tyrosine kinase that is distinguished by a unique extracellular domain homologous to the lectin Discoidin I found Dictyostelium discoideum. A cosmid was isolated from a human chromosome 6 cosmid library containing the DDR gene. A complete genomic contig of the DDR gene was constructed from seven subclones of the cosmid. The cosmid fragments were analyzed by PCR, sequencing, and comparison of genomic/cDNA sequence. The DDR gene is composed of 17 exons, ranging in size from 96 to 1014 bp, distributed along approximately 12 kb of genomic DNA. The extracellular domain is encoded by 8 exons of which three code for the discoidin domain. The transmembrane domain is encoded by 1 exon, the juxtamembrane by 3 exons, and the catalytic domain by 5 exons. The generation of the two splice variants of DDR, EDDR1 and EDDR2 are explained by the genomic structure. Exon 11 (111 bp in the juxtamembrane domain) is present in DDR and absent in the splice variant EDDR1. An inverted repeat of 20 bp was identified at the 3' exon-intron junction of exon 11, which results in a lariat loop-like secondary structure. EDDR2 is generated because of a cryptic splice acceptor site that results in an extra 18 bp (6 amino acids) inserted 5' of exon 14 in the catalytic domain. A polymorphic (GT)17 repeat was identified in intron 5 with a heterozygosity of 0.71. The exon-intron structure of the DDR gene will be helpful in further understanding of its function and explains the possible structural basis for the two splice variants.


Subject(s)
Chromosome Mapping/methods , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Mitogen/genetics , Alternative Splicing , Cell Membrane , Chromosomes, Human, Pair 6/genetics , DNA, Complementary/genetics , Dinucleotide Repeats/genetics , Discoidin Domain Receptors , Exons/genetics , Gene Library , Genes/genetics , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Polymerase Chain Reaction/methods , RNA/chemistry , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid
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