33 results on '"Johnson, Gibbes R."'
Search Results
2. Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors
- Author
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Madhavarao, Chikkathur N., Agarabi, Cyrus D., Wong, Lily, Müller-Loennies, Sven, Braulke, Thomas, Khan, Mansoor, Anderson, Howard, and Johnson, Gibbes R.
- Published
- 2014
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3. The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors
- Author
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Wu, Chuan-Jin, O'Rourke, Donald M, Feng, Gen-Sheng, Johnson, Gibbes R, Wang, Qiang, and Greene, Mark I
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- 2001
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4. Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line
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Ma, Lin, Gauvillé, Christiane, Berthois, Yolande, Millot, Guy, Johnson, Gibbes R, and Calvo, Fabien
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- 1999
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5. Expression and localization of five members of the testis-specific serine kinase (Tssk) family in mouse and human sperm and testis
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Li, Yahui, Sosnik, Julian, Brassard, Laura, Reese, Michael, Spiridonov, Nikolay A., Bates, Tonya C., Johnson, Gibbes R., Anguita, Juan, Visconti, Pablo E., and Salicioni, Ana M.
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- 2011
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6. Use of Analytical Ultracentrifugation as an Orthogonal Method for Size Exclusion Chromatography: Assuring Quality for Therapeutic Protein Products and Meeting Regulatory Expectations.
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Carpenter, John F., Bain, David L., and Johnson, Gibbes R.
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- 2016
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7. Identification of N-Glycans Displaying Mannose-6-Phosphate and their Site of Attachment on Therapeutic Enzymes for Lysosomal Storage Disorder Treatment.
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Bones, Jonathan, Mittermayr, Stefan, McLoughlin, Niaobh, Hihiard, Mark, Wynne, Kieran, Johnson, Gibbes R., Grubb, Jeffrey H., Sly, William S., and Rudd, Pauline M.
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- 2011
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8. Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates.
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Ogurtsov, Aleksey Y., Mariño-Ramírez, Leonardo, Johnson, Gibbes R., Landsman, David, Shabalina, Svetlana A., and Spiridonov, Nikolay A.
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GENE expression ,GENETIC regulation ,GENETIC transcription ,PROTEIN kinases ,BIOLOGICAL evolution ,GENOMES ,PHOSPHORYLATION ,BIOLOGICAL variation ,GENETIC mutation - Abstract
Background: Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood. Principal Findings: Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly. Conclusions/Significance: PK genomic architecture, the size of gene functional domains and evolutionary rates correlate with the pattern of gene expression. Structure and evolutionary divergence of tissue-specific PK genes is related to the proliferative activity of the tissue where these genes are predominantly expressed. Our data provide evidence that physiological requirements for transcription intensity, ubiquitous expression, and tissue-specific regulation shape gene structure and affect rates of evolution. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
9. Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA.
- Author
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Chakrabarti, Kausik, Rong Lin, Schiller, Noraisha I., Yanping Wang, Koubi, David, Ying-Xin Fan, Rudkin, Brian B., Johnson, Gibbes R., and Schiller, Martin R.
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RHO GTPases ,CELLULAR signal transduction ,G proteins ,NERVE growth factor ,PROTEIN-tyrosine kinases ,CELL receptors ,NEURONS ,PROTEIN binding - Abstract
Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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10. Identification and Characterization of SSTK, a Serine/Threonine Protein Kinase Essential for Male Fertility.
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Spiridonov, Nikolay A., Wong, Lily, Zerfas, Patricia M., Starost, Matthew F., Pack, Svetlana D., Paweletz, Cloud P., and Johnson, Gibbes R.
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SERINE proteinases ,PROTEIN kinases ,HEAT shock proteins ,SPERMATOGENESIS ,HUMAN fertility ,CHROMATIN - Abstract
Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly consented among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1β, HSC7O, and HSP7O proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones Hi, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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11. Distinct Domains in the SHP-2 Phosphatase Differentially Regulate Epidermal Growth Factor Receptor/NF-κB Activation through Gab1 in Glioblastoma Cells.
- Author
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Kapoor, Gurpreet S., Yi Zhan, Gurpreet S., Johnson, Gibbes R., and O'Rourke, Donald M.
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PHOSPHATASES ,EPIDERMAL growth factor ,GLIOBLASTOMA multiforme ,CELLS ,TRANSCRIPTION factors ,NF-kappa B - Abstract
The transcription factor nuclear factor κB (NF-κB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-κB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-κB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-κB activation. We also show that EGFR-induced NF-κB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-κB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-κB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-κB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-κB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-κB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-κB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-κB activation. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
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12. Expression of epidermal growth factor-related proteins in the aged adult mouse mammary gland and their relationship to tumorigenesis.
- Author
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Herrington, Eugene E., Ram, Tracy G., Salomon, David S., Johnson, Gibbes R., Gullick, William J., Kenney, Nicholas, and Hosick, Howard L.
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- 1997
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13. Characterization of a novel amphiregulin-related molecule in 12- O-tetradecanoylphorbol-13-acetate-treated breast cancer cells.
- Author
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Martinez-Lacaci, Isabel, Johnson, Gibbes R., Salomon, David S., and Dickson, Robert B.
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- 1996
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14. Expression of Amphiregulin, a Novel Gene of the Epidermal Growth Factor Family, in Human Gastric Carcinomas.
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Kitadai, Yasuhiko, Yasui, Wataru, Yokozaki, Hiroshi, Kuniyasu, Hiroki, Ayhan, Ayse, Haruma, Ken, Kajiyama, Goro, Johnson, Gibbes R., and Tahara, Eiichi
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- 1993
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15. Frequent Immunohistochemical Detection of EGF Supergene Family Members in Ovarian Carcinogenesis.
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Stromberg, Kurt, Johnson, Gibbes R., O'Connor, Dennis M., Sorensen, Craig M., Gullick, William J., and Kannan, Bhanu
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- 1994
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16. Amphiregulin antisense oligonucleotide inhibits the growth of T3M4 human pancreatic cancer cells and sensitizes the cells to EGF receptor-targeted therapy.
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Funatomi, Hitoshi, Itakura, Jun, Ishiwata, Toshiyuki, Pastan, Ira, Thompson, Stewart A., Johnson, Gibbes R., and Korc, Murray
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- 1997
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17. Association of Epidermal Growth Factor-related Peptides and Type I Receptor Tyrosine Kinase Receptors with Prognosis of Human Colorectal Carcinomas.
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Saeki, Toshiaki, Salomon, David S., Johnson, Gibbes R., Gullick, Willium J., Mandai, Koichi, Yamagam, Keitaro, Moriwaki, Shosuke, Tanada, Minoru, Takashima, Shigemitsu, and Tahara, Eiichi
- Published
- 1995
18. TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis.
- Author
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Jha, Kula N., Tripurani, Swamy K., and Johnson, Gibbes R.
- Subjects
PROTAMINES ,GENE silencing ,SPERMATOGENESIS - Abstract
Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
19. Tssk6 is required for Izumo relocalization and gamete fusion in the mouse.
- Author
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Sosnik, Julian, Miranda, Patricia V., Spiridonov, Nikolay A., Sook-Young Yoon, Fissore, Rafael A., Johnson, Gibbes R., and Visconti, Pablo E.
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FERTILIZATION in vitro ,SPERMATOZOA ,GAMETES ,ACTIN ,CLINICAL trials - Abstract
One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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20. The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry
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Bieber, Allan L., Becker, Robert R., McParland, Reginald, Hunt, Donald F., Shabanowitz, Jeffrey, Yates, John R., III, Martino, Paul A., and Johnson, Gibbes R.
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- 1990
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21. Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea
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Johnson, Gibbes R. and Bieber, Allan L.
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- 1988
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22. Heat Shock Protein 90 Functions to Stabilize and Activate the Testis-specific Serine/Threonine Kinases, a Family of Kinases Essential for Male Fertility.
- Author
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Jha, Kula N., Coleman, Alyssa R., Wong, Lily, Salicioni, Ana M., Howcroft, Elizabeth, and Johnson, Gibbes R.
- Subjects
- *
HEAT shock proteins , *TESTIS , *THREONINE , *KINASES , *HAPLOIDY - Abstract
Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
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23. Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants.
- Author
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Ying-Xin Fan, Lily Wong, Marino, Michael P., Wu Ou, Yi Shen, Wen Jin Wu, Kwok-Kin Wong, Reiser, Jakob, and Johnson, Gibbes R.
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LUNG cancer , *PHOSPHORYLATION , *TRANSGENIC mice , *CELL lines , *TYROSINE - Abstract
Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2YVMA and ERBB2G776VC, have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2YVMA to wild type using physiologically relevant peptide substrates reveals that ERBB2YVMA kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by α150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2YVMA phosphorylates GAB1 protein α70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutantinduced oncogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
24. Identification of a Novel HSP70-binding Cochaperone Critical to HSP90-mediated Activation of Small Serine/Threonine Kinase.
- Author
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Jha, Kula N., Lily Wong, Zerfas, Patricia M., de Silva, Rukman S., Ying-Xin Fan, Spiridonov, Nikolay A., and Johnson, Gibbes R.
- Subjects
- *
GERM cells , *FERTILITY , *ZONA pellucida , *POLYPEPTIDES , *CYTOPLASM - Abstract
We previously reported the identification of small serine/ threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (Kd ~10 nM), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldana- mycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaper- one that is critical to the HSP90-mediated activation of SSTK. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
25. Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism.
- Author
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Ying-Xin Fan, Wong, Lily, Jinhui Ding, Spiridonov, Nikolay A., Johnson, Richard C., and Johnson, Gibbes R.
- Subjects
- *
GENETIC mutation , *GENETICS , *PROTEIN kinases , *CYCLIN-dependent kinases , *PHOSPHOTRANSFERASES , *GLYCOGEN synthase kinase-3 - Abstract
Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the αC helix and β4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to ∼10- and ∼7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the αC-β4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the αC-β4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the αC-β4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of αC-β4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
26. Ligand Regulates Epidermal Growth Factor Receptor Kinase Specificity.
- Author
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Ying-Xin Fan, Wong, Lily, Deb, Tushar B., and Johnson, Gibbes R.
- Subjects
- *
EPIDERMAL growth factor , *PROTEINS , *PEPTIDES , *CYTOKINES , *PHOSPHORYLATION , *TYROSINE , *AMINO acid sequence - Abstract
The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, Km and kcat, revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR- mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased heat ∼5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of ∼5-fold in the specificity constants (kcat/Km) for EGFR peptides, whereas ∼15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr- 317 sites in purified GST fusion proteins ∼4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
27. Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development.
- Author
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Tripurani SK, Wang Y, Fan YX, Rahimi M, Wong L, Lee MH, Starost MF, Rubin JS, and Johnson GR
- Subjects
- Animals, Apoptosis drug effects, Cell Count, Cell Differentiation drug effects, Cell Proliferation drug effects, DNA Damage, Gene Deletion, Gene Expression Regulation, Developmental drug effects, Gene Knock-In Techniques, Hair growth & development, Hair Follicle cytology, Ligands, Mice, Mitosis drug effects, Models, Biological, Morphogenesis drug effects, Phenotype, RNA, Messenger genetics, RNA, Messenger metabolism, Skin growth & development, Transcription, Genetic drug effects, Transforming Growth Factor alpha pharmacology, ras Proteins metabolism, ErbB Receptors metabolism, Hair Follicle growth & development, Hair Follicle metabolism, Wnt Signaling Pathway drug effects
- Abstract
Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.
- Published
- 2018
- Full Text
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28. Acquired substrate preference for GAB1 protein bestows transforming activity to ERBB2 kinase lung cancer mutants.
- Author
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Fan YX, Wong L, Marino MP, Ou W, Shen Y, Wu WJ, Wong KK, Reiser J, and Johnson GR
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adenosine Triphosphate genetics, Adenosine Triphosphate metabolism, Amino Acid Substitution, Animals, Antineoplastic Agents pharmacology, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic pathology, Female, Humans, Lapatinib, Lung Neoplasms genetics, Lung Neoplasms pathology, Mice, Mice, Nude, Mice, Transgenic, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphoproteins genetics, Phosphorylation genetics, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Quinazolines pharmacology, Receptor, ErbB-2 genetics, Signal Transduction drug effects, Signal Transduction genetics, Substrate Specificity drug effects, Substrate Specificity genetics, Adaptor Proteins, Signal Transducing metabolism, Cell Transformation, Neoplastic metabolism, Lung Neoplasms metabolism, Mutation, Missense, Phosphoproteins metabolism, Receptor, ErbB-2 metabolism
- Abstract
Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2(YVMA) and ERBB2(G776VC), have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2(YVMA) to wild type using physiologically relevant peptide substrates reveals that ERBB2(YVMA) kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2(YVMA) phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis.
- Published
- 2013
- Full Text
- View/download PDF
29. Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.
- Author
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Fan YX, Wong L, Ding J, Spiridonov NA, Johnson RC, and Johnson GR
- Subjects
- Adenosine Triphosphate metabolism, Animals, COS Cells, Catalysis, Chlorocebus aethiops, Enzyme Activation, ErbB Receptors metabolism, HSP90 Heat-Shock Proteins metabolism, Humans, Kinetics, Models, Molecular, Peptides metabolism, Protein Binding, Protein Structure, Tertiary, Receptor, ErbB-2 chemistry, Signal Transduction, Substrate Specificity, Mutation genetics, Receptor, ErbB-2 antagonists & inhibitors, Receptor, ErbB-2 metabolism
- Abstract
Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.
- Published
- 2008
- Full Text
- View/download PDF
30. EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity.
- Author
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Fan YX, Wong L, and Johnson GR
- Subjects
- Amino Acid Sequence, Catalysis, Epidermal Growth Factor metabolism, Humans, Kinetics, Ligands, Molecular Sequence Data, Multigene Family, Phosphorylation, Protein Binding, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Catalytic Domain, ErbB Receptors metabolism, Genes, erbB physiology
- Abstract
We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279 , 38143-38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k(cat) approx. 5-fold, but does not significantly alter the K(m) for any ErbB peptides. Furthermore, peptides containing lysine at position -2 or -3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K(m) and increased the k(cat) for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.
- Published
- 2005
- Full Text
- View/download PDF
31. Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites.
- Author
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Fan YX, Wong L, Deb TB, and Johnson GR
- Subjects
- Animals, Binding Sites, Blotting, Western, COS Cells, Catalytic Domain, Cell Line, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Enzyme Activation, Epidermal Growth Factor metabolism, Epitopes chemistry, Glutathione Transferase metabolism, Kinetics, Ligands, Mice, Peptides chemistry, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Protein-Tyrosine Kinases metabolism, Recombinant Fusion Proteins chemistry, Shc Signaling Adaptor Proteins, Src Homology 2 Domain-Containing, Transforming Protein 1, Substrate Specificity, Time Factors, Tyrosine chemistry, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport metabolism, ErbB Receptors metabolism, Phosphoproteins metabolism
- Abstract
The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.
- Published
- 2004
- Full Text
- View/download PDF
32. Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor receptor/NF-kappaB activation through Gab1 in glioblastoma cells.
- Author
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Kapoor GS, Zhan Y, Johnson GR, and O'Rourke DM
- Subjects
- Adaptor Proteins, Signal Transducing, Base Sequence, Cell Line, Tumor, DNA, Neoplasm genetics, ErbB Receptors genetics, Glioblastoma genetics, Humans, Intracellular Signaling Peptides and Proteins, Models, Biological, Mutagenesis, Site-Directed, NF-kappa B genetics, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphoproteins genetics, Phosphorylation, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatases genetics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, Recombinant Proteins genetics, Recombinant Proteins metabolism, SH2 Domain-Containing Protein Tyrosine Phosphatases, Signal Transduction, src Homology Domains, ErbB Receptors metabolism, Glioblastoma metabolism, NF-kappa B metabolism, Phosphoproteins metabolism, Protein Serine-Threonine Kinases, Protein Tyrosine Phosphatases chemistry, Protein Tyrosine Phosphatases metabolism
- Abstract
The transcription factor nuclear factor kappaB (NF-kappaB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-kappaB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-kappaB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-kappaB activation. We also show that EGFR-induced NF-kappaB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-kappaB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-kappaB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-kappaB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-kappaB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-kappaB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-kappaB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-kappaB activation.
- Published
- 2004
- Full Text
- View/download PDF
33. Transforming growth factor alpha, amphiregulin and cripto-1 are frequently expressed in advanced human ovarian carcinomas.
- Author
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D'Antonio A, Losito S, Pignata S, Grassi M, Perrone F, De Luca A, Tambaro R, Bianco C, Gullick WJ, Johnson GR, Iaffaioli VR, Salomon DS, and Normanno N
- Subjects
- Adult, Aged, Amphiregulin, EGF Family of Proteins, Female, GPI-Linked Proteins, Glycoproteins genetics, Humans, Immunohistochemistry, Intercellular Signaling Peptides and Proteins genetics, Middle Aged, Neoplasm Proteins genetics, Ovarian Neoplasms etiology, Ovarian Neoplasms mortality, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Transforming Growth Factor alpha genetics, Epidermal Growth Factor, Glycoproteins analysis, Intercellular Signaling Peptides and Proteins analysis, Membrane Glycoproteins, Neoplasm Proteins analysis, Ovarian Neoplasms chemistry, Transforming Growth Factor alpha analysis
- Abstract
The expression of transforming growth factor alpha (TGFalpha), amphiregulin (AR) and cripto-1 (CR-1) was assessed by immunohistochemistry in 83 specimens (59 primary ovarian tumors and 24 extra-ovarian carcinomas) that were obtained from 68 ovarian carcinoma patients. Within the 59 primary tumors, 54 (92%) expressed immunoreactive TGFalpha, 45 (76%) expressed AR, and 28 (47%) expressed CR-1. The expression of AR and CR-1 mRNAs in the ovarian carcinomas was also demonstrated by RT-PCR analysis. Seventeen extra-ovarian specimens (71%) were found to express CR-1, whereas AR and TGFalpha were expressed respectively in 21 (87%) and 22 (92%) extra-ovarian tissues. In 15 cases for whom both ovarian and extra-ovarian tissues were available, a statistically significant higher expression of CR-1 was found in extra-ovarian specimens. A statistically significant correlation was found between AR expression in the ovarian carcinomas and both low grade and low proliferative activity. Finally, expression of TGFalpha was predictive of longer progression-free survival. These data strongly suggest that the EGF-related peptides might be involved in the pathogenesis and outcome of human ovarian cancer.
- Published
- 2002
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