Cloning of a Grb2 Isoform with Apoptotic Properties

Abstract

Growth factor receptor-bound protein 2 (Grb2) links tyrosine-phosphorylated proteins to a guanine nucleotide releasing factor of the son of sevenless (Sos) class by attaching to the former by its Src homology 2 (SH2) moiety and to the latter by its SH3 domains. An isoform of grb2 complementary DNA (cDNA) was cloned that has a deletion in the SH2 domain. The protein encoded by this cDNA, Grb3-3, did not bind to phosphorylated epidermal growth factor receptor (EGFR) but retained functional SH3 domains and inhibited EGF-induced transactivation of a Ras-responsive element. The messenger RNA encoding Grb3-3 was expressed in high amounts in the thymus of rats at an age when massive negative selection of thymocytes occurs. Microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced apoptosis. These findings indicate that Grb3-3, by acting as a dominant negative protein over Grb2 and by suppressing proliferative signals, may trigger active programmed cell death.

In bone marrow derived Baf-3 cells, inhibition of apoptosis by IL-3 is mediated by two independent pathways

“…These results complement with previously published data showing that activated Ras could induce the Bcl-X gene (Kinoshita et al, 1995b) and demonstrate that the MAP-kinase pathway is critical for the apoptosis inhibition. The Ras/MAP-kinase pathway has been shown to be involved in the regulation of cell survival in a number of systems (Fath et al, 1994;Kinoshita et al, 1995a;Swan et al, 1995). For example MAP-kinase plays a key role in the inhibition of cell death which takes place following positive selection of T cells in the thymus .…”

Expression of a novel isoform of Vav, Vav-T, containing a single Src homology 3 domain in murine testicular germ cells

“…We detected coexpression of hnRNPK and vav-T transcripts in the testicular germ cells. In the case of Grb3-3, a naturally occurring isoform of Grb2 that lacks functional SH2 domain but retains functional SH3 domains, it is supposed to act as a dominant negative protein for Grb2 (Fath et al, 1994). Vav-T is, however, unlikely to function as an inhibitor of Vav in the male germ cells, since no coexpression was observed in them.…”

Induction of Apoptosis in Human Pancreatic Carcinoma Cells by a Synthetic Bleomycin‐like Ligand

“…The DNA samples were subjected to electrophoresis in a 1.5% agarose gel and visualized by ethidium bromide staining. Flow-cytometric analysis Cells (2×10 6 ) were seeded in 10-cm culture dishes. After 24 h, chemicals were added to the cells, and the cultures were incubated for the desired periods.…”

“…In cultured cells, apoptosis is often induced by activation or inhibition of specific signaling pathways. Activation of FAS on the cell surface by anti-FAS, 1) addition of tumor necrosis factor-α (TNF-α) 2) or transforming growth factor-β1 (TGF-β1), 3) inhibition of tyrosine kinase, 4,5) and intracellular injection of an inactive Grb 2 isoform 6) were reported to induce apoptosis. Characteristic features of apoptosis include cell shrinkage, apoptotic body formation, internucleosomal DNA fragmentation, and early induction of cell death after the signal transduction.…”