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Biomedical subjects

H J Kung

Publications and source records attributed to H J Kung.

At least 19 recordsLinked to original sources
PubMed2002

Molecular indications for in vivo integration of the avian leukosis virus, subgroup J-long terminal repeat into the Marek's disease virus in experimentally dually-infected chickens.

Marek's disease virus, a herpesvirus, and avian leukosis virus, subgroup J, a retrovirus, are oncogenic viruses of poultry. Both viruses may infect the same flock, the same bird and the same cell. In a double-infected cell, the retroviral DNA can integrate into the cellular or the Marek's disease virus (MDV) genome. The retroviral-long terminal repeat (LTR) integration into MDV was first described by Isfort et al., (Proc Natl Acad Sci 89, 991-995, 1992) following tissue culture co-infection. The recombinant virus isolated, RM1, had altered biological properties compared to the parental MDV (Witter R.L., Li D., Jones D., and Kung H.-J., Avian Dis 41, 407-421, 1997) . The issue of retroviral sequence integration into herpesviruses in vivo, in cases of double-virus infection is of wide significance in general virology and veterinary medicine; it also represents a special case of gene transposition. Using the avian system, we aimed to determine occurrence of such integrations in vivo. Chickens were experimentally co-infected with both avian leukosis virus (ALV) subgroup J and with MDV. To demonstrate the presence of the retroviral LTR in the MDV genome we applied the Hot Spot-combined PCR assay (Borenshtain R. and Davidson I., J Virol Meth 82, 119-127, 1999) that consisted of two consecutive steps of amplification. By that HS-cPCR assay, certain MDV genomic sites, defined as HS for integration were specifically amplified, the HS step, and then subjected to screening in an attempt to detect LTR inserts. The screening was achieved by amplification using heterologous primer sets, one for the MDV hot spot and the other for the retroviral LTR, the cPCR step. The products were Southern blotted and hybridized with MDV and ALV-LTR probes. Chimeric molecules were detected and evidenced by an intense signal in 3/10 chickens and weakly in other 3/10 birds. Detection was by LTR amplification, sequencing and multiple alignment to the ALV-J-LTR sequence. The present study indicated that chimeric molecules were produced in vivo.

Animals↗
PubMed2001

MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulating ERK pathway.

In this study, we report the cloning and characterization of a novel human Ste20-related kinase that we designated MST4. The 416 amino acid full-length MST4 contains an amino-terminal kinase domain, which is highly homologous to MST3 and SOK, and a unique carboxy-terminal domain. Northern blot analysis indicated that MST4 is highly expressed in placenta, thymus, and peripheral blood leukocytes. Wild-type but not kinase-dead MST4 can phosphorylate myelin basic protein in an in vitro kinase assay. MST4 specifically activates ERK but not JNK or p38 MAPK in transient transfected cells or in stable cell lines. Overexpression of dominant negative MEK1 or treatment with PD98059 abolishes MST4-induced ERK activity, whereas dominant-negative Ras or c-Raf-1 mutants failed to do so, indicating MST4 activates MEK1/ERK via a Ras/Raf-1 independent pathway. HeLa and Phoenix cell lines overexpressing wild-type, but not kinase-dead, MST4 exhibit increased growth rate and form aggressive soft-agar colonies. These phenotypes can be inhibited by PD98059. These results provide the first evidence that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation.

Amino Acid Sequence↗
PubMed2001

Growth hormone interacts with the Marek's disease virus SORF2 protein and is associated with disease resistance in chicken.

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by a herpesvirus, the MD virus (MDV). Because MD is a significant economic problem to the poultry industry, there is great interest in enhancing genetic resistance, which is controlled by multiple genes. The influence of the MHC has been clearly demonstrated, and several relevant quantitative trait loci have been mapped; however, no single gene influencing MD resistance has been identified. Transcription of SORF2 is perturbed in the MDV recombinant clone RM1 due to a solo insertion of the reticuloendotheliosis virus long terminal repeat, which may explain the loss of oncogenicity for this strain. Hypothesizing that SORF2-interacting host proteins are involved in MD resistance, we screened a chicken splenic cDNA library by the yeast two-hybrid assay using SORF2 as bait. The chicken growth hormone (GH) structural peptide was identified, and the specific interaction was verified by coimmunoprecipitation. Immunohistochemical staining and indirect immunofluorescence assay indicated that GH and SORF2 can be coexpressed in MDV-infected cells both in vitro and in vivo. Furthermore, polymorphism in the GH gene (GH1) is associated with the number of tissues with tumors in commercial White Leghorn chickens with the MHC B*2/B*15 genotype. We conclude that GH1 may well be a MD resistance gene.

Animals↗
PubMed2001

Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells.

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

Animals↗
PubMed2001

Proteolytic activation of ETK/Bmx tyrosine kinase by caspases.

Etk/Bmx is a member of the Btk/Tec family of kinases, which are characterized by having a pleckstrin homology domain at the N terminus, in addition to the Src homology 3 (SH3), SH2, and the catalytic domains, shared with the Src family kinases. Etk, or Btk kinases in general, has been implicated in the regulation of apoptosis. To test whether Etk is the substrate for caspases during apoptosis, in vitro translated [(35)S]methionine-labeled Etk was incubated with different apoptotic extracts and recombinant caspases, respectively. Results showed that Etk was proteolyzed in all conditions tested with identical cleavage patterns. Caspase-mediated cleavage of Etk generated a C-terminal fragment, containing the complete SH2 and tyrosine kinase domains, but without intact pleckstrin homology and SH3 domains. This fragment has 4-fold higher kinase activity than that of the full-length Etk. Ectopic expression of the C-terminal fragment of Etk sensitized the PC3 prostate cancer cells to apoptosis in response to apoptosis-inducing stimuli. The finding, together with an earlier report that Etk is potentially antiapoptotic, suggests that Etk may serve as an apoptotic switch, depending on the forms of Etk existing inside the cells. To our knowledge, this is the first case where the activity of a tyrosine kinase is induced by caspase cleavage.

Apoptosis↗
PubMed2001

Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain.

Etk/BMX, a member of the Btk family of tyrosine kinases, is highly expressed in cells with great migratory potential, including endothelial cells and metastatic carcinoma cell lines. Here, we present evidence that Etk is involved in integrin signalling and promotes cell migration. The activation of Etk by extracellular matrix proteins is regulated by FAK through an interaction between the PH domain of Etk and the FERM domain of FAK. The lack of Etk activation by extracellular matrix in FAK-null cells could be restored by co-transfection with wild-type FAK. Disrupting the interaction between Etk and FAK diminished the cell migration promoted by either kinase. Furthermore, inhibiting Etk expression in metastatic carcinoma cell lines with an antisense oligonucleotide blocks integrin-mediated migration of these cells. Taken together, our data indicate the essential role of the interaction of the PH domain of Etk and the FERM domain of FAK in integrin signalling.

Animals↗
PubMed2001

Hierarchical cleavage of focal adhesion kinase by caspases alters signal transduction during apoptosis of intestinal epithelial cells.

BACKGROUND & AIMS: Purified intestinal epithelial cells die of detachment-induced apoptosis due to loss of cell anchorage during isolation. Anchorage-dependent cells form focal adhesions, sites of enhanced cell-matrix attachment that confer survival signals. Focal adhesion kinase (FAK), a component of the focal adhesion signaling complex, transduces these antiapoptotic signals. In this report, the molecular events leading to cleavage of FAK by caspases during apoptosis and its functional implications are defined. METHODS: Cytosolic extracts of human intestinal epithelial cells undergoing detachment-induced apoptosis were analyzed by Western blotting, immunoprecipitation, and kinase assay. RESULTS: FAK is cleaved by the ordered proteolytic activity of 2 different members of the caspase-3 family. The first cleavage is mediated by caspase-3, generating a 94/92-kilodalton-terminal fragment, which is processed by caspase-6 to an 84-kilodalton fragment. After apoptosis is initiated, the level of FAK phosphorylation is rapidly decreased, and the phosphorylation pattern of FAK-associated proteins is dramatically modified, showing significant yet divergent changes in signal transduction. CONCLUSIONS: Cleavage of FAK during apoptosis of normal human cells is an example of the sequential, highly regulated, and coordinate action of caspases that not only dismantle a cell by proteolysis, but also alter the cell's signaling machinery.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Marek's disease virus (MDV) encodes an interleukin-8 homolog (vIL-8): characterization of the vIL-8 protein and a vIL-8 deletion mutant MDV.

Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to the BamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (gamma2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8DeltasmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8DeltasmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8DeltasmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.

Amino Acid Sequence↗
PubMed2001

Identification of protein kinases dysregulated in CD4(+) T cells in pathogenic versus apathogenic simian immunodeficiency virus infection.

Human immunodeficiency virus infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM) leads to a generalized loss of immune responses involving perturbations in T-cell receptor (TCR) signaling. In contrast, naturally SIV-infected sooty mangabeys (SM) remain asymptomatic and retain immune responses despite relatively high viral loads. However, SIV infection in both RM and SM led to similar decreases in TCR-induced Lck phosphorylation. In this study, a protein tyrosine kinase (PTK) differential display method was utilized to characterize the effects of in vivo SIV infection on key signaling molecules of the CD4(+) T-cell signaling pathways. The CD4(+) T cells from SIV-infected RM, but not SIV-infected SM, showed chronic downregulation of baseline expression of MLK3, PRK, and GSK3, and symptomatically SIV-infected RM showed similar downregulation of MKK3. In vitro TCR stimulation with or without CD28 costimulation of CD4(+) T cells did not lead to the enhancement of gene transcription of these PTKs. While the CD4(+) T cells from SIV-infected RM showed a significant increase of the baseline and anti-TCR-mediated ROR2 transcription, SIV infection in SM led to substantially decreased anti-TCR-stimulated ROR2 transcription. TCR stimulation of CD4(+) T cells from SIV-infected RM (but not SIV-infected SM) led to the repression of CaMKKbeta and the induction of gene transcription of MLK2. Studies of the function of these molecules in T-cell signaling may lead to the identification of potential targets for specific intervention, leading to the restoration of T-cell responses.

Animals↗
PubMed2001

Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase.

The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth inhibition caused by androgen ablation. (ii) Tyrosine kinases are involved in neurotrophic factor-mediated AR activation and, as such, may serve as targets of future therapeutics, to be used in conjunction with current antihormone and antineuropeptide therapies.

Androgens↗
PubMed2001

MCF-10A-NeoST: a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.

PURPOSE: There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. EXPERIMENTAL DESIGN: We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. RESULTS: NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in three-dimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, and EphA2 tyrosine kinases in transformed MCF-10A cells. CONCLUSIONS: MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.

Breast Neoplasms↗
PubMed2001

Retroviral insertion into herpesviruses: discovery and implications.

Retroviruses and herpesviruses are important human pathogens. Co-infections of these viruses in hosts often exist and co-infection of the same cell type is also common. In this report, we will review the evidence that supports the contention that retrovirus insertion into herpesvirus can occur in vitro and in vivo. We will also discuss retrovirus insertion into other large DNA viruses.

Herpesviridae↗
PubMed2000

Protein-tyrosine phosphatase D1, a potential regulator and effector for Tec family kinases.

Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain. The signaling mechanisms regulating Etk kinase activity remain largely unknown. To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen. Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated. The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin. In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction. Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction. Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors. The Etk-PTPD1 interaction also increased Stat3 activation. The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation. In addition, Tec (but not Btk) kinase can also be activated by PTPD1. Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.

Animals↗
PubMed2000

Tyrosine kinases and gastric cancer.

Carcinoma of the stomach is one of the most prevalent cancer types in the world today. Two major forms of gastric cancer are distinguished according to their morphological and clinicopathological classifications (well differentiated/intestinal type and poorly differentiated/diffuse type), characteristics that could also be attributed to the altered expression of different types of oncogenes or tumor suppressor genes. Significant differences exist for gastric cancer incidence comparing people of different ethnic origins, implicating various genetic and epigenetic factors for gastric oncogenesis. There are only a limited number of molecular markers available for gastric cancer detection and prognostic evaluation, among which are tyrosine kinases. There is convincing evidence that tyrosine kinases are involved in oncogenesis and disease progression for many human cancers. Amplifications of certain tyrosine kinases (c-met, k-sam and erbB2/neu) have been associated with human gastric cancer progression. Alternatively spliced transcripts and enhanced protein-expression levels for some of these tyrosine kinases are correlated with clinical outcomes for gastric cancer patients. With advent of high throughput techniques, it is now possible to detect nearly all expressed tyrosine kinases in a single screen. This increases the chance to identify additional tyrosine kinases as predictive markers for gastric cancers. In this article, we will first review the literature data concerning certain tyrosine kinases implicated in gastric carcinogenesis and then summarize more recent work which provide comprehensive tyrosine kinase profiles for gastric cancer specimens and cell lines. Two new gastric cancer molecular markers (tie-1 and mkk4) have been identified through the use of these profiles and demonstrated effective as clinical prognostic indicators.

Humans↗
PubMed2000

Signaling network of the Btk family kinases.

The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus.

We have determined the DNA sequence of the unique long (UL) region and the repeat long (RL) region in the genome of serotype 1 GA strain of Marek's disease virus (MDV), a member of the alpha-herpesvirus family. With this information, the complete nucleotide sequence of GA-MDV is now known. The entire GA-MDV genome is predicted to be about 174 kbp in size, with an organization of TRL-UL-IRL-IRS-US-TRS, typical of a alpha-herpesvirus. The UL sequence contains 113,508 bp and has a base composition of 41.7% G + C. A total of 67 ORFs were identified completely within the UL region, among which 55 are homologous to genes encoded by herpes simplex virus-1. Twelve of them are unique with presently unknown functions. The sequence of RL reported here together with those published earlier reveal the major structural features of the RL. Virtually all of the ORFs encoded by RL are specific to serotype I of MDV. These ORFs are likely to contribute to some of the unique biological properties of MDV. Among the proteins encoded by MDV-specific ORFs are Meq, a jun/fos family of transcriptional factor implicated in transformation and latency, virus-encoded interleukin-8, a CXC chemokine, and pp38 and pp24, two phosphoproteins with undefined functions. There is also a putative lipase gene (LORF2) that has homologies in HPRS-24 (serotype II) strain of MDV and in various avian adenoviruses. An additional unique feature of MDV is the presence of long terminal repeat remnant sequences of avian retrovirus reticuloendotheliosis virus. These remnant sequences are derived from the U3-enhancer region through ancestral insertions by reticuloendotheliosis virus proviruses.

DNA Replication↗
PubMed2000

KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

Amino Acid Sequence↗