Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Rasinduced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.Phosphoinositide 3-kinases (PI3K) 1 are enzymes that transfer phosphate to position 3 of the phosphoinositide ring, regulating a variety of cell responses including survival, division, and transformation. PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only the class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (3-poly-PtdIns) products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor (GFR) stimulation (for a review, see Refs. 1-4). 3-Poly-PtdIns recruits pleckstrin homology domain-containing proteins such as phosphoinositide-dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (5-8).Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, of which there are several isoforms (1, 2, 9 -14). The prototypic p85 regulatory subunit has an SH3 domain, a BcR homology domain (BH) flanked by proline-rich sequences, and two SH2 domains separated by the so-called inter-SH2 domain, to which p110 binds (15-17). The p110 catalytic subunit contains a p85-interacting region, a Ras-binding domain, a region homologous to PI4K and the PI3K catalytic domain (4, 15). The p85 subunit protects p110 from degradation and inhibits its enzymatic activity in quiescent cells (18). When cells are stimulated by receptors with intrinsic Tyr kinase activity, such as platelet-derived growth factor receptor (PDGFR), p85 mediates p110 translocation to the cell membrane, where PI3K substrates are found (11). In addition to mediating PI3K translocation, p85 appears to transmit an activating conformational change to p110 (19 -21), because binding to p85 of Tyr-phosphorylated peptides representing the activated PDGFR increases p110 enzymatic activity (16,22). Other receptors activate PI3K by stimulating cytosolic Tyr ki...

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT-mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor-deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl-3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S-phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl-3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.

Stability and copy number of extra‐chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid‐free cells. Instead, we demonstrate here that Kid becomes active in plasmid‐containing cells when plasmid copy number decreases, cleaving not only host‐ but also a specific plasmid‐encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection.

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.