gdae Yun

HBx protein of hepatitis B virus activates Jak1-STAT signaling

The X-gene product (HBx) of the hepatitis B virus plays essential roles in viral replication and the generation of hepatocellular carcinoma. Although the mechanism for HBx action is unclear, HBx may exert its pleiotropic functions through the stimulation of signal transduction pathways including the Ras/mitogen-activated protein kinase cascade and/or inactivation of the p53 function. Here, we investigated whether HBx has the ability to activate the Jak-STAT signaling pathway. As a first step, we established stable cell lines constitutively expressing HBx. In these HBx-expressing stable cells, the tyrosine phosphorylation of various STATs, including STAT3 and -5, was constitutively enhanced by HBx, and the concomitant increase in STAT-dependent DNA binding and transcriptional activation was observed. Furthermore, HBx specifically elevated tyrosine phosphorylation and in vitro kinase activity of Jak1, but not Jak2 or Tyk2, through protein to protein interaction with Jak1. These results clearly establish HBx as the inducer of the Jak-STAT signaling pathway, and at the same time, HBx-mediated Jak-STAT activation may provide a novel mechanism for the pleiotropic functions of HBx, including transformation and promiscuous transcriptional activation.

X-gene Product of Hepatitis B Virus Induces Apoptosis in Liver Cells

Hepatitis B virus is a causative agent of hepatocellular carcinoma, and in the course of tumorigenesis, the X-gene product (HBx) is known to play important roles. Here, we investigated the transforming potential of HBx by conventional focus formation assay in NIH3T3 cells. Cells were cotransfected with the HBx expression plasmid along with other oncogenes including Ha-ras, v-src, v-myc, v-fos, and E1a. Unexpectedly, the introduction of HBx completely abrogated the focus-forming ability of all five tested oncogenes. In addition, the cotransfection of Bcl-2, an apoptosis inhibitor, reversed the HBx-mediated inhibition of focus formation, suggesting that the observed repression of focus formation by HBx is through the induction of apoptosis. Next, to test unequivocally whether HBx induces apoptosis in liver cells, we established stable Chang liver cell lines expressing HBx under the control of a tetracycline-inducible promoter. Induction of HBx in these cells in the presence of 1% calf serum resulted in typical apoptosis phenomena such as DNA fragmentation, nuclear condensation, and fragmentation. Based on these results, we propose that HBx sensitizes liver cells to apoptosis upon hepatitis B virus infection, contributing to the development of hepatitis and the subsequent generation of hepatocellular carcinoma.

LIME, a Novel Transmembrane Adaptor Protein, Associates with p56lck and Mediates T Cell Activation

In this study, we identify and characterize a novel transmembrane adaptor protein, designated Lck-interacting membrane protein (LIME), as a binding partner of the Lck Src homology (SH)2 domain. LIME possesses a short extracellular domain, a transmembrane domain, and a cytoplasmic tail containing five tyrosine-based motifs. The protein is primarily expressed in hematopoietic cells and lung. Interestingly, LIME expression is up-regulated by TCR stimulation and sustained up to 24 h, suggesting that LIME acts throughout the early to late stages of T cell activation. LIME is localized to membrane rafts and distributed within the T cell–APC contact site. Upon TCR stimulation of Jurkat T cells, LIME associates with Lck as a tyrosine-phosphorylated protein. Experiments using Jurkat T cells expressing CD8–LIME chimera reveal that the protein associates with phosphatidylinositol 3-kinase, Grb2, Gads, and SHP2, and activates ERK1/2 and JNK but not p38. Moreover, overexpression of LIME in Jurkat T cells induces transcriptional activation of the IL-2 promoter. Our data collectively show that LIME is a raft-associated transmembrane adaptor protein linking TCR stimuli to downstream signaling pathways via associations with Lck.

Rosmarinic Acid Induces p56lck-Dependent Apoptosis in Jurkat and Peripheral T Cells via Mitochondrial Pathway Independent from Fas/Fas Ligand Interaction

Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56lck (Lck)-dependent manner; Lck+ Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck− Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck+ subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA’s ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.

Rosmarinic acid inhibits TCR‐induced T cell activation and proliferation in an Lck‐dependent manner

Lck is a T cell‐restricted Src family protein tyrosine kinase that plays pivotal roles in TCR‐mediated signaling. We aimed to identify novel agents that could disrupt the molecular interactionof the Src homology 2‐domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Large‐scale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR‐induced‐Ca2+ mobilization and IL‐2 promoter activation but not phorbol 12‐myristate 13‐acetate/ionomycin‐induced IL‐2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR‐induced splenocyte proliferation as well as one‐way MLR at an IC50 of 25–50 μM and inhibited cytokine expression such as IL‐2 and IFN‐γ. Here, we first report RosA as an inhibitor of TCR‐signaling and subsequent T cell proliferation.

SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation

SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDA-MB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ER-mediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogen-induced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-type-specific manner and act as a novel transcriptional co-regulator in ER signalling.

SOCS-6 negatively regulates T cell activation through targeting p56LCK to proteasomal degradation

The T cell-specific tyrosine kinase, p56lck, plays crucial roles in T cell receptor (TCR)-mediated T cell activation. Here, we report that SOCS-6 (suppressor of cytokine signaling-6) is a negative regulator of p56lck. SOCS-6 was identified as a protein binding to the kinase domain of p56lck through yeast two-hybrid screening. SOCS-6 bound specifically to p56lck (F505), which mimics the active form of p56lck, but not to wild type p56lck. In Jurkat T cells, SOCS-6 binding to p56lck was detected 1–2 h after TCR stimulation. Confocal microscopy showed that upon APC-T cell conjugation, SOCS-6 was recruited to the immunological synapse and colocalized with the active form of p56lck. SOCS-6 promoted p56lck ubiquitination and its subsequent targeting to the proteasome. Moreover, SOCS-6 overexpression led to repression of TCR-dependent interleukin-2 promoter activity. These results establish that SOCS-6 acts as a negative regulator of T cell activation by promoting ubiquitin-dependent proteolysis.

Liver-specific Enhancer II Is the Target for the p53-mediated Inhibition of Hepatitis B Viral Gene Expression

Here, we established the inhibitory mechanism of p53 on hepatitis B viral gene expression using HepG2 cells. Our results are as follows. First, p53 down-regulated the activities of all four promoters of hepatitis B virus (HBV), suggestive of the presence of a common element mediating the p53-dependent transcriptional repression. Second, employing the 5′-deletion constructs of the pregenomic/core promoter, the liver-specific enhancer II region was localized as a target for the p53-mediated transcriptional repression. Third, in a detailed analysis of the enhancer II region, the 5′-proximal 31-base pair region was defined as a p53-repressible element. Throughout the study, p53-mediated repression was rescued upon coexpression of the X-gene product, HBx. Finally, in an electrophoretic mobility shift assay, the defined p53-repressible element did not bind purified p53 directly, but shifted three bands in HepG2 nuclear extract, two of which was supershifted upon addition of p53 monoclonal antibody. These results display a novel mechanism of p53-dependent transcriptional repression in which p53 negatively regulates the viral-specific DNA enhancer through protein to protein interaction with an enhancer-binding protein. At the same time, the results indicate that p53 plays a defensive role against HBV by transcriptionally repressing the HBV core promoter through liver-specific enhancer II and HBx is required to counteract this inhibitory function of p53.

Direct Interaction of the CD38 Cytoplasmic Tail and the Lck SH2 Domain CD38 TRANSDUCES T CELL ACTIVATION SIGNALS THROUGH ASSOCIATED Lck

CD38 ligation has been shown to induce activation of intracellular signaling cascade in T lymphocytes through a Lck-dependent pathway. However, it is not clear how Lck initiates the CD38-mediated signaling process. In the present study, we showed that CD38 and Lck were physically associated through the cytoplasmic tail and the Src homology 2 domain, respectively. This was evidenced by coimmunoprecipitation of Lck with CD38 and Lck with isolated CD38 cytoplasmic domain from T cell lysate, cell lysate of COS-7 cells cotransfected with cDNAs of Lck and CD38, or a mixture of in vitro translated CD38 and Lck. Because the CD38 cytoplasmic domain does not contain any tyrosine residue, the interaction should be independent of phosphotyrosine. The interaction was further confirmed by in vitro interaction between a purified Lck Src homology 2 domain and a nonphosphosynthetic peptide corresponding to the membrane proximal region of the CD38 cytoplasmic domain. In addition, CD38 ligation resulted in an elevated tyrosine kinase activity of the CD38-associated Lck and ultimate activation of interleukin-2 gene transcription. Furthermore, expression of a kinase-deficient Lck mutant suppressed interleukin-2 gene activation in a dose-dependent manner. These results strongly suggested that CD38 ligation indeed tranduced signals for T cell activation using its associated Lck.

The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein.

The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin α6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration.