Cytokinin-induced expression of OSH1 in a shoot-regenerating rice callus.

The expression of a KNOX class 1 gene OSH1 is induced by cytokinin during regeneration of shoots from callus in Oryza sativa L. (rice). This cytokinin-induced expression was enhanced by overexpression of homologues of cytokinin-signalling phosphorelay genes such as a histidine kinase gene OHK3, a phosphotransmitter gene OHP2 and a response regulator gene ORR1 in cultured cells. Regionally overlapped expression of these genes and OSH1 was observed in shoot apex. These results suggest that these cytokinin-signalling genes are positive regulators of the expression of OSH1, and mediate the OSH expression upon shoot regeneration from callus in rice.

Novel CD3-specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLCγ1 following T-cell stimulation.

The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4(+) T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3ε Ab, 145-2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-γ1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.

Abl kinase interacts with and phosphorylates vinexin.

Non-receptor tyrosine kinase Abl is a well known regulator of the actin-cytoskeleton, including the formation of stress fibers and membrane ruffles. Vinexin is an adapter protein consisting of three SH3 domains, and involved in signal transduction and the reorganization of actin cytoskeleton. In this study, we found that vinexin alpha as well as beta interacts with c-Abl mainly through the third SH3 domain, and that vinexin and c-Abl were colocalized at membrane ruffles in rat astrocytes. This interaction was reduced by latrunculin B, suggesting an F-actin-mediated regulatory mechanism. We also found that vinexin alpha but not beta was phosphorylated at tyrosine residue when c-Abl or v-Abl was co-expressed. A mutational analysis identified tyrosine 127 on vinexin alpha as a major site of phosphorylation by c- or v-Abl. These results suggest that vinexin alpha is a novel substrate for Abl.

Role of interaction with vinculin in recruitment of vinexins to focal adhesions.

Although vinexin was originally identified as a protein binding to the proline-rich hinge region of vinculin, the functions and biochemical properties of the vinexin-vinculin interaction are not known. Here, we determined the affinity of the vinexin-vinculin interaction using surface plasmon resonance measurements and found that vinexin beta interacts with the C-terminal half of vinculin, which mimics an activated "open" form, with a threefold higher affinity than with the full-length "closed" vinculin. Coimmunoprecipitation experiments showed that cell adhesion on fibronectin enhances the vinexin-vinculin interaction. We also show that the interaction with vinculin is necessary for the efficient localization of vinexin alpha and beta at focal adhesions. These observations suggest a model that "activated" vinculin localized at focal adhesions recruits vinexins to focal adhesions.