Showing papers by "Hiroyuki Mano published in 1998"

Tec/Bmx non‐receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Gα12/13

Abstract: A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase

Abstract: Background Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. Results One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. Conclusion Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Abstract: Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets

Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts.

Abstract: We investigated the feasibility of an inducible apoptosis system to regulate cells genetically engineered for ectopic cytokine production. In a previous study, cDNA encoding the ligand-binding domain of the rat estrogen receptor was fused to the sequence for murine Fas transmembrane and cytoplasmic regions, and expression of the fusion protein (MfasER) in L929 fibroblasts resulted in estrogen-dependent apoptosis. We applied this MfasER/estrogen strategy to apoptosis-mediated regulation of cytokine production, using the human granulocyte colony-stimulating factor (G-CSF) as a model. Upon estrogen treatment, the G-CSF producers expressing MfasER showed an apoptotic phenotype and died in several hours, with termination of G-CSF production. This estrogen-induced apoptosis was not influenced by whether the target cells were proliferating or resting, unlike a conventional suicide system involving the herpes simplex virus 1 thymidine kinase (HSVtk). That is, estrogen induced prompt and extensive apoptosis in the resting cells which expressed MfasER, while ganciclovir treatment induced only partial reduction of the resting cells which expressed HSVtk. These results imply the feasibility of apoptosis-mediated regulation of cytokine production by genetically modified cells for supplement gene therapy.