Masaki Shirakata

Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia.

Dok-3 sequesters Grb2 and inhibits the Ras-Erk pathway downstream of protein-tyrosine kinases.

Adaptor proteins are essential in coordinating recruitment and, in a few cases, restraint of various effectors during cellular signaling. Dok‐1, Dok‐2 and Dok‐3 comprise a closely related family of adaptor, which negatively regulates mitogen‐activated protein kinase Erk downstream of protein‐tyrosine kinases (PTKs). Recruitment of p120 rasGAP, a potent inhibitor of Ras, by Dok‐1 and Dok‐2 appears critical in the negative regulation of the Ras‐Erk pathway. However, as Dok‐3 does not bind rasGAP, it has been unclear how Dok‐3 inhibits Erk downstream of PTKs. Here, we identified Grb2 as a Dok‐3‐binding protein upon its tyrosine phosphorylation. This interaction required the intact binding motifs of the Grb2 SH2 domain, and a mutant (Dok‐3‐FF) having a Tyr/Phe substitution at these motifs failed to inhibit Ras and Erk activation downstream of a cytoplasmic PTK Src. Because Grb2 forms a stable complex with Sos, a crucial activator of Ras, these data suggest that Dok‐3 restrains Grb2 and inhibits the ability of the Grb2‐Sos complex to activate Ras. Indeed, forced expression of Dok‐3, but not Dok‐3‐FF, inhibited the recruitment of the Grb2‐Sos complex to Shc downstream of Src, which is an essential event for activation of the Ras‐Erk pathway. These findings indicate that Dok‐3 sequesters Grb2 from Shc and inhibits the Ras‐Erk pathway downstream of PTKs.

CD40 ligand is a critical effector of Epstein–Barr virus in host cell survival and transformation

Epstein–Barr virus (EBV), implicated in numerous human diseases, including lymphoid malignancies, persistently infects peripheral B cells and transforms them into lymphoblastoid cell lines. Here we found that EBV equally infected B cells from patients with X-linked hyper IgM syndrome and those from healthy donors; however, it hardly transformed X-linked hyper IgM syndrome B cells, because of the dysfunctional gene of CD40 ligand (CD40L) of the patients. Unlike CD40, CD40L is not usually expressed on B cells. However, we found that EBV infection of normal B cells induced CD40L expression as a critical effector in host cell transformation and survival. Moreover, chronic active EBV infection of peripheral T cells, implicated in T cell malignancies, was associated with ectopic expression of CD40, and, in Jurkat T cells, EBV infection induced CD40 expression. These results suggest that EBV infection induces CD40L/CD40 signaling in host cells, which appears to play an essential role in its persistent infection and malignancies of lymphocytes.

Replication Licensing of the EBV oriP Minichromosome

The latent EBV genome may persist in the integrated form as well as the circular episomal form. However, most of the latent viral DNA molecules are known to exist in the circular episomal form, which binds to host chromosomes during mitosis. The DS element of oriP in the circular episomal DNA functions as a replication origin. As it replicates once in a single S phase, it is possible that oriP is regulated by the cellular replication licensing mechanism including the MCM family of replication licensing factors. Transient replication analysis using the oriP plasmid and HeLa/EB1 cells revealed that the DS element requires early G1 phase for the next round of replication, the same cell-cycle window in which the replication licensing of cellular chromatin occurs. After this phase, the sedimentation velocity of the oriP minichromosome increases. MCM2 associates with the oriP minichromosome at late G1 but not at G2/M, and this association requires the DS element in the plasmid. The interaction of EBNA1 and the MCM proteins on the DS element was also suggested. These results suggested that the cellular licensing mechanism controls the replication from oriP. This also suggested a similarity in the replication machinery of the cellular chromatin and the latent EBV genome. In addition to DS-dependent replication, the EBV genome replicates in a manner independent of the DS element in several cultured cell lines. The DS-dependent replication is likely to be suppressed in these cell lines by the expression of other viral proteins. In contrast, EBV-positive Burkitts lymphoma and circulating EBV-infected B cells express only EBNA1 or both EBNA1 and LMP2. DS-dependent replication may play a major role in these EBNA1-only cells, and the licensing regulation of oriP is important for maintenance of the EBV genome during this latent period of the viral life cycle. EBNA1 is required for efficient nuclear retention and partitioning of oriP-carrying plasmid by its binding to the FR element, thus providing stable persistence of the latent EBV genome during cell division. The copy number of latent EBV DNA molecules in B-cell lines remains fairly constant during multiple passage in culture. However, very little is known about the mechanism by which the viral DNA molecules are equally segregated into daughter cells. To understand the mechanisms responsible for stable nuclear retention and partitioning of the latent viral genome, it is essential to analyze the episomal and integrated viral DNAs at a single-cell level by FISH and other techniques.

The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers.

Summary. In cell lines established from Marek’s disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek’s Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

Novel Immediate-Early Protein IE19 of Human Cytomegalovirus Activates the Origin Recognition Complex I Promoter in a Cooperative Manner with IE72

ABSTRACT The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) expresses IE86, IE72, IE55, and IE18 mRNA by differential splicing. Reverse transcription-PCR with IE72-specific primers generated an 0.65-kb cDNA from HCMV-infected fibroblast RNA, which does not correspond to any known MIE cDNA. Nucleotide sequencing revealed that the 0.65-kb cDNA is from exons 1, 2, and 3 and part of exon 4, indicating that it is derived from a novel alternatively spliced mRNA of the MIE gene. The cDNA encodes a 172-amino-acid polypeptide, termed IE19, which corresponds to an IE72 variant with an internal deletion from Val86 to Pro404 and appears as a band at 38 kDa on a sodium dodecyl sulfate-polyacrylamide gel. IE19 mRNA was expressed at a low level in the immediate-early, early, and late period of viral infection. IE19 was localized in nuclei, and a transient-expression assay revealed that IE19 enhances IE72-dependent activation of the HsOrc1 promoter, which is identified here as an IE72 target promoter. Another MIE protein, IE86, activated the same promoter but only weakly compared to IE72, and coexpression of IE19 did not alter the IE86-mediated transcriptional activation. In addition, IE19 did not enhance the IE72-dependent activation of the HCMV UL54 promoter. These results suggest that IE19 is a transcriptional coactivator that works with IE72.

Activation of TRAF5 and TRAF6 Signal Cascades Negatively Regulates the Latent Replication Origin of Epstein-Barr Virus through p38 Mitogen-Activated Protein Kinase

ABSTRACT Latent Epstein-Barr virus (EBV) is maintained by the virus replication origin oriP that initiates DNA replication with the viral oriP-binding factor EBNA1. However, it is not known whether oriPs replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressed oriP activity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressed oriPactivity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-inducedoriP suppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitts lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level of oriP activity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.

Placental Extravillous Cytotrophoblasts Persistently Express Class I Major Histocompatibility Complex Molecules after Human Cytomegalovirus Infection

ABSTRACT Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.

Epstein-Barr virus (EBV)-infected cells were frequently but dispersely detected in T-cell lymphomas of various types by in situ hybridization with an RNA probe specific to EBV-specific nuclear antigen 1.

Association of Epstein-Barr virus (EBV) with T-cell lymphomas was examined by in situ hybridization (ISH) with an antisense probe specific to abundantly expressed EBV-encoded small RNA-1 (EBER1). In addition to EBER1, EBV-specific nuclear antigen-1 (EBNA-1) is commonly expressed in EBV-associated tumors and latently infected B-lymphocytes. We examined paraffin sections of T-cell lymphomas except those of nasal origin for expression of latent viral transcripts by ISH. Using ISH with improved antisense RNA probe specific to EBNA-1 mRNA, the virus was detected in 19 (59%) of 32 cases, whereas the EBER1 transcript was found in only 15 (47%) of 32 cases by conventional EBER-ISH, resulting in 21 EBV-positive cases (66%) by combining the two methods. Latent membrane protein 1 (LMP1) mRNA of EBV was detected in 15 of 32 cases (47%), while no EBNA2 expression was observed in any these tumors. Patients with these lymphomas positive for LMP1 expression showed lower survival rates than those without expression of the viral mRNA. These results indicate that, in addition to EBER-ISH, RNA-RNA ISH with EBNA1 probes could be useful for detection of EBV-infected cells in paraffin sections, and detection of LMP1 mRNA expression in tumor cells could be a useful prognostic factor for T-cell lymphoma.

Tumorigenicity of mouse BALB/c 3T3 fibroblast cells which express Epstein-Barr virus-encoded LMP1 and show normal growth phenotypes in vitro is correlated with loss of transforming growth factor-β1-mediated growth inhibition

SummaryLatent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) genome is known to induce loss of contact inhibition and the anchorage-independent growth in rodent fibroblasts and increased expression of cell-surface activation markers and cell adhesion molecules in human B lymphocytes. To analyze the role of LMP1 in tumorigenicity, we prepared BALB/c 3T3 clones (B3LP) expressing LMP1. These B3LP cells showed non-transformed phenotypes in vitro which were characterized by normal cell morphology, contact inhibition in growth and anchorage-dependent growth. The activity of NF-κB induced generally in several cell lines after transfer of the LMP1 gene was not detected in B3LP cells. However, B3LP expressing LMP1 at moderate levels lost sensitivity to growth arrest by transforming growth factor-β1(TGF-β1) and formed tumors in severe combined immune deficiency mice. Cells expressing the truncated form of LMP1 and expressing LMP1 at low level were sensitive to TGF-β1-mediated growth arrest and did not form tumors in mice. Therefore, cells expressing LMP1 at moderate but not at low levels formed tumors in mice and lost sensitivity to TGF-β1. Our results suggest that loss of TGF-β1-mediated growth inhibition is an important event for the tumorigenicity of LMP1-expressing cells.