Masaho Ishino

CLONING AND CHARACTERIZATION OF CELL ADHESION KINASE BETA , A NOVEL PROTEIN-TYROSINE KINASE OF THE FOCAL ADHESION KINASE SUBFAMILY

A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase β (CAKβ), was identified by cDNA cloning. The rat CAKβ is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAKβ has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAKβ is a protein structurally related to but different from FAK. The CAKβ gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAKβ antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAKβ cDNA. The tyrosine-phosphorylated state of CAKβ was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAKβ localized to sites of cell-to-cell contact in COS-7 transfected with CAKβ cDNA, in which FAK was found at the bottom of the cells. Thus, CAKβ is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.

Mutational Analysis of HIV-1 Tat Minimal Domain Peptides: Identification of Trans-Dominant Mutants That Suppress HIV-LTR-Driven Gene Expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.

CELL ADHESION KINASE BETA FORMS A COMPLEX WITH A NEW MEMBER, HIC-5, OF PROTEINS LOCALIZED AT FOCAL ADHESIONS

Cell adhesion kinase β (CAKβ/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKβ-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor β1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitrowith the extreme C-terminal region (residue 801 to the end) of CAKβ. CAKβ was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKβ with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKβ. Coimmunoprecipitation of Hic-5 with CAKβ, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKβ and Myc-tagged Hic-5, was lost when the CAKβ amino acid residues 741–903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKβ was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKβ, implying possible involvement of Hic-5 in the downstream signaling of CAKβ.

Phylogenetic analysis of rotaviruses with genotypes G1, G2, G9 and G12 in Bangladesh: evidence for a close relationship between rotaviruses from children and adults

To clarify the phylogenetic relatedness of rotaviruses causing gastroenteritis in children and adults, an epidemiologic investigation was conducted in Mymensingh, Bangladesh, during the period between July 2004 and June 2006. A total of 2,540 stool specimens from diarrheal patients from three hospitals were analyzed. Overall, rotavirus-positive rates in children and adults were 26.4 and 10.1%, respectively. Among the 155 rotavirus specimens examined genetically from both children and adults, the most frequent G genotype was G2 (detection rate: 54.0 and 47.6%, respectively), followed by G1 (21.2 and 26.2%, respectively), and G9 (15.9 and 9.5%, respectively). G12 was also detected in five specimens (3.2% in total; four children and one adult). Sequence identities of VP7 genes of G2 rotaviruses from children and adults were higher than 97.8%, while these Bangladeshi G2 viruses showed generally lower identities to G2 rotaviruses reported elsewhere in the world, except for some strains reported in African countries. Similarly, extremely high sequence identities between children and adults were observed for VP7 genes of G1, G9 and G12 rotaviruses, and also for the VP4 genes of P[4], P[6], and P[8] viruses. Rotaviruses from children and adults detected in this study were included in a single cluster in phylogenetic dendrograms of VP7 or VP4 genes of individual G/P types. Rotaviruses with two emerging types, G9 and G12, had VP7 genes that were phylogenetically close to those of individual G-types recently reported in Bangladesh and India and were included in the globally spreading lineages of these G-types. These findings suggested that genetically identical rotaviruses, including those with the emerging types G9 and G12, were circulating among children and adults in city and rural areas of Bangladesh.

Whole genomic characterization of a human rotavirus strain B219 belonging to a novel group of the genus rotavirus

Novel rotavirus strains B219 and ADRV‐N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3. In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined. The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes. The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV‐N (strain J19) sequences (87.7–94.3% and 88.7–98.7%, respectively). In contrast, sequence identity to groups A–C rotavirus genes was less than 61%. However, functionally important domains and structural characteristics in VP1‐VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins. Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups. J. Med. Virol. 80:2023–2033, 2008.

Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

Interleukin-2 induces tyrosine phosphorylation of SHP-2 through IL-2 receptor beta chain.

Coupling of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) induces rapid increase in tyrosine phosphorylation of cellular substrates through activation of non-receptor protein tyrosine kinases. Here, we report that stimulation through the IL-2R induced tyrosine phosphorylation of the SH2-containing protein-tyrosine phosphatase SHP-2 in F7, a hematopoietic BAF-B03 transfectant clone expressing the IL-2Rβ chain. The tyrosine phosphorylation of SHP-2 was specific since another protein-tyrosine phosphatase SHP-1, which is structurally homologous to SHP-2, was not tyrosine phosphorylated. The IL-2-induced tyrosine phosphorylation of SHP-2 required the acidic region within the IL-2Rβ chain where Src-family PTKs interact. Though the serine-rich region within IL-2Rβ chain was also required for the phosphorylation of SHP-2, Jak3 activation was dispensable. In COS-7 cells, co-expression of SHP-2 with Lyn resulted in increased tyrosine phosphorylation levels of SHP-2, whereas co-expression of SHP-2 with Fyn failed to alter the levels significantly. Considering that Lyn and Fyn are major Src-family PTKs expressed in BAF-B03 cells, our data suggest that Lyn may be principally responsible for the tyrosine phosphorylation of SHP-2 in F7 cells. Furthermore, the IL-2 stimulation also induced tyrosine phosphorylation of SHP-2 in the human IL-2-dependent T-cell line ILT-Mat. Taken together, these studies demonstrate an involvement of SHP-2 in the IL-2-mediated signaling events through the activation of specific PTKs.

Phylogenetic Analysis of Rotaviruses With Predominant G3 and Emerging G9 Genotypes From Adults and Children in Wuhan, China

Prevalence and phylogenetic relatedness of rotaviruses causing diarrheal diseases in children and adults were analyzed in Wuhan, China. During a period between June 2006 and February 2008, group A rotavirus was identified in 24.9% (280/1126) and 7.6% (83/1088) of specimens taken from children and adults, respectively. G3P[8] was the most frequent genotype in both children (66.3%) and adults (62.7%), followed by G1P[8] (20.3% and 26.2%, respectively). G9 was detected in specimens from six children (2.0%) and seven adults (5.6%). The VP7 genes of G3P[8] rotaviruses from children and adults showed extremely high sequence identities to each other (98.9–100%) and also to those of G3 viruses isolated in Wuhan in 2003–2004. In the phylogenetic analysis of the VP7 gene, the G3P[8] rotaviruses in Wuhan were clustered into a single lineage with some G3 viruses, which had been referred to as “the new variant G3” rotaviruses, reported recently in East Asia and Southeast Asia. Similar to G3P[8] rotaviruses, extremely high sequence identities between children and adults were observed for VP7 genes of G1 and G9 rotaviruses. The G9 viruses were clustered in the lineage of globally spreading strains, while G1 viruses were genetically close to those reported previously in China and Japan. These findings indicated the persistence of the variant G3 rotaviruses and spread of G9 rotaviruses derived from the global G9 lineage in Wuhan, and suggested that the rotaviruses were circulating among children and adults, irrelevant to the G types. J. Med. Virol. 81:382–389, 2009.

Genetic analysis of an ADRV-N-like novel rotavirus strain B219 detected in a sporadic case of adult diarrhea in Bangladesh

Summary.An unusual human rotavirus strain B219 was detected in a stool specimen from a 65-year old patient with diarrhea in Bangladesh during April 2002. Cloning and sequence analysis of five genes of the B219 strain indicated that this virus is genetically closely related to the ADRV-N strain, which caused an adult diarrhea outbreak in China, but distinct from groups A, B, and C rotaviruses known to cause diarrheal diseases in humans. Accordingly, rotavirus strains B219 and ADRV-N were considered to belong to a novel group of human rotavirus, and the ADRV-N-like novel human rotaviruses were suggested to be distributed to a geographically wider area.

Whole-genome characterization of human group C rotaviruses: identification of two lineages in the VP3 gene.

Group C rotavirus (GCRV) is distributed worldwide as an enteric pathogen in humans and animals. However, to date, whole-genome sequences are available only for a human strain (Bristol) and a porcine strain (Cowden). To investigate the genetic diversity of human GCRVs, nearly full-length sequences of all 11 RNA segments were determined for human GCRVs detected recently in India (v508), Bangladesh (BS347), China (Wu82 and YNR001) and Japan (OH567 and BK0830) and analysed phylogenetically with sequence data for GCRVs published previously. All the RNA segments of human GCRV strains except for the VP3 gene showed high levels of conservation (>93 % nucleotide sequence identity, >92 % amino acid sequence identity), belonging to a single genetic cluster distinct from those of animal GCRVs. In contrast, the VP3 genes of human GCRVs could be discriminated into two clusters, designated M2 and M3, that were distinguished phylogenetically from those of porcine and bovine GCRVs (clusters M1 and M4, respectively). Between M2 and M3, amino acid sequence identity of the VP3 gene was 84.1-84.7 %, whereas high identities were observed within each cluster (92.3-97.6 % for M2, 98.2-99.3 % for M3). Sequence divergence among the four VP3 clusters was observed throughout the amino acid sequence except for conserved motifs, including those possibly related to enzyme functions of VP3. The presence of obvious genetic diversity only in the VP3 gene among human GCRVs suggested that either the M2 or M3 VP3 gene of human GCRVs might have been derived through reassortment from an animal GCRV or from an unidentified human GCRV strain belonging to a novel genogroup.