A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase ␤ (CAK␤), was identified by cDNA cloning. The rat CAK␤ 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 CAK␤ 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 CAK␤ is a protein structurally related to but different from FAK. The CAK␤ gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK␤ antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK␤ cDNA. The tyrosinephosphorylated state of CAK␤ was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK␤ localized to sites of cell-tocell contact in COS-7 transfected with CAK␤ cDNA, in which FAK was found at the bottom of the cells. Thus, CAK␤ is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts. Protein-tyrosine kinases (PTKs)1 that do not span the plasma membranes (so-called nonreceptor PTKs) have been classified into different subclasses (subfamilies) based on the sequence similarity and distinct structural characteristics (1). Many nonreceptor PTKs participate in cellular signal transduction by associating with the intracellular portions of transmembrane receptors which do not themselves have PTK activity. Different nonreceptor PTKs play diverse and specific roles in mediating the signal transduction by different nonkinase receptors (2-4).Focal adhesion kinase (FAK) has been proposed as the prototype (and hitherto the sole member) of a new subfamily of nonreceptor PTK, represented by proteins with large N-and C-terminal domains flanking the catalytic domain but without Src homology 2 and 3 (SH-2 and SH-3) domains (5-9). FAK is concentrated in focal adhesions (5, 6), and its phosphorylation and activation are triggered by the ligand binding to integrins and by the stimulation of certain growth factor and neuropeptide receptors (6, 10 -24). The N-and C-terminal domains of FAK mediate its interactions with integrins, the Src-family kinases and paxillin, a focal adhesion associated protein (8,9,(25)(26)(27)(28). By these and other yet to be characterized interactions, FAK regulates signaling via different receptors. Because only one member of the FAK subfamily is known to date, we sought to identify a second PTK of the FAK subfamily by a homologybased cDNA cloning strategy. We describe here an isolation and characterization of a cDNA coding for a new member of the FAK family. The novel PTK described here is the second member, to our knowledge, of the FAK subfamily whose cDNA has been cloned and sequenced and is designated CAK␤ for cell adhesion kinase ␤.

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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.

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. INTRODUCTIONRotavirus, a member of the family Reoviridae, is the most important viral pathogen that causes gastroenteritis in humans. The rotavirus genome consists of 11 segments of dsRNA, and the viral particle is composed of three concentric layers, the outer capsid, inner capsid and core (Estes & Kapikian, 2007). The outer capsid consists of two structural proteins, VP4 and VP7, which contain neutralization antigens. The inner capsid consists of structural protein VP6. Rotavirus is classified into seven groups, A-G, based on the antigenicity of the inner capsid protein VP6 and genomic characteristics (Kapikian et al., 2001). In humans, groups A, B and C have been detected to date. Group A rotavirus (GARV) is the most prevalent throughout the world and is recognized as the leading viral pathogen of acute gastroenteritis in children. For epidemiological investigations of GARV, a genetic classification system based on the outer capsid proteins VP7 (G type) and VP4 (P type) has been adopted (Santos & Hoshino, 2005). In addition, a full-genome-based genotyping system composed of genotypes of the 11 individual RNA segments has been proposed on the basis of full-The GenBank/EMBL/DDBJ accession numbers for the GCRV sequences determined in this study are HQ185629-HQ185631 (v508), HQ185632-HQ185642 (BS347), HQ185643-HQ185651 (Wu82), HQ185652-HQ185662 (YNR001), HQ185663-HQ185672 (OH567) and ...

Efs was originally found by expression cloning of a mouse embryo cDNA library through its Fyn-SH3 binding capacity (Ishino et al., Oncogene 11, 2331± 2338. Efs has characteristic regions important in intracellular signal transduction; these are an SH3 domain, a cluster of putative ligands for SH2 domains and proline-rich sequences with SH3-binding consensus. In this paper, we report cDNA cloning of human Efs and a variant of it from a hippocampal cDNA library. The human Efs gene was mapped to chromosome 14q11.2-q12 by¯uorescence in situ hybridization. We identi®ed two forms of human Efs, designated hEfs1 and hEfs2. hEfs1 represents the human counterpart of original mouse embryo Efs (mEfs1). hEfs2, the newly identi®ed form, is identical to hEfs1, except for its lack of the SH3 domain. hEfs1 and mEfs1 are 80% identical in their amino acid sequences and 100% identical within the SH3 domain. Reverse transcription polymerase chain reaction analysis of adult mouse tissue RNA indicated expression of Efs2 and of Efs1 in various tissues. Evidence suggesting the presence of the Efs2 protein in human tissue was obtained by immunoprecipitation followed by immunoblotting with two di erent anti-Efs antibodies. Possible functions of Efs2 are discussed.

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein.In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.CrkL is a Src homology (SH)2 1 and SH3 adapter (1-3). Through its SH2 domain, CrkL binds to focal adhesions proteins like paxillin and Cas (1-3). CrkL also binds to a Rapspecific guanine nucleotide exchange factor, C3G, through its N-terminal SH3 domain and, thus, conveys C3G to focal adhesions. C3G activates a small GTPase Rap1 and regulates cell adhesion and spreading, indicating that the CrkL-C3G complex is a critical component of focal adhesions (4). We previously reported that CrkL is present in human platelets, and that it is an adapter for WASP, syk, or phosphorylated STAT5 (5-7).On the other hand, ADP-ribosylation factors (Arfs) are also members of the Ras-related small GTPases and function in the regulation of membrane trafficking and actin cytoskeleton (8, 9). Similar to other GTPases, the activity of Arfs is regulated positively by GEFs and negatively by GTPase-activating proteins (GAPs). Recently, several Arf GAPs have been cloned and characterized and found to have phosphoinositide-dependent GAP activity toward Arfs (10). ASAP1 (also called DEF-1, for differentiation enhancing factor-1), the prototype of the phosphoinositide-dependent Arf GAP family, is a multidomain protein with pleckstrin homology, Arf GAP, ankyrin repeat, proline-rich region, and SH3 domains. ASAP1 binds to phosphatidylinositol (4, 5)P 2 through its PH domain and shows GAP activity toward Arf (11-13). In NIH3T3 cells, endogenous ASAP1 localizes in focal adhesions and, when overexpressed, ASAP1 affects cell spreading of NIH3T3 cells on fibronectin (14). Although the function of Arf in focal adhesions is not clear yet, the localization of ASAP1 and its effect on cell spreading suggests the importance of Arf signaling on the dynamics of focal adhesions.During our continual efforts to clarify the role of CrkL in the regulation of signal transduction, we found that the SH3 domain of CrkL binds to ASAP1. The data obtained from studies using platelets and COS7 cells overexpressing ASAP1 revealed that CrkL is a critical lynchpin between ASAP1 and focal adhesions. EXPERIMENTAL PROCEDURESBlo...

ABSTRACT. Cell adhesion kinase β (CAKβ/PYK2) is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAKβ localizes at the perinuclear region in fibroblasts. Here we expressed in cultured cells two point mutants of CAKβ, P717A and P859A, each of which had lost one of its two PXXP motifs, the ligand sequence for SH3 domains, found at the CAKβ C-terminal region. We observed a remarkable change in the subcellular distribution of the P859A mutant; while that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKβ also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAKβ shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKβ, a CAKβ-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKβ and co-expressed Hic-5 formed nuclear speckles, in which one other CAKβ-binding protein, p130Cas , was also concentrated. These findings on nuclear translocation of CAKβ imply that CAKβ may regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.