Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex

Studi sulla persistenza dei sister choromatid exchanges, spontanei e indotti, attraverso tre cicli cellulari, a livello di singola cellula

Dottorato di ricerca in scienze genetiche. 8. ciclo. A.a. 1994-95. Tutore R. Barale. Coordinatore I. BarraiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

The nature of high frequency sister chromatid exchange cells (HFCs)

We employed the three-way differential staining technique (TWD), which allows SCEs to be distinguished on a per generation basis by scoring third metaphases (M3), in order to study the spontaneous levels of SCEs in normal and high frequency cells (HFCs) that occurred in the first (S1), second (S2) and third (S3) S phases. Fifty one of 900 lymphocytes from 37 healthy donors were defined as HFCs by calculating the 95th percentile of the distribution of SCEs in S1 + S2. 'Normal' cells presented almost the same number of SCEs after the first, second and third cell cycles (SCE averages of 2.43, 2.04 and 3.53 respectively). In contrast, HFCs showed a higher SCE count in S1, which decreased rapidly through the cycles and reached baseline level at S3 (SCE averages of 7.18, 4.29 and 3.45 respectively). This would suggest that the lesions responsible for the higher SCE frequency in HFCs were effectively removed after two cell cycles and strongly support the hypothesis that HFCs are lymphocytes which accumulate higher levels of DNA lesions through time

Modulating factors of individual sensitivity to diepoxybutane: chromosome aberrations induced in vitro in human lymphocytes

Peripheral blood lymphocytes from a sample of 62 randomly selected donors were analysed for spontaneous and diepoxybutane (DEB)-induced chromosomal aberrations (CA). These individuals were part of a larger sample of 122 subjects whose DEB responsiveness was evaluated by means of sister chromatid exchange (SCE) analysis. Confounding factors (such as smoking, wine and coffee consumption, occupation and haematological factors) were analysed for their effect on individual DEB-responsiveness, but no statistically significant associations were observed. Interestingly, a bimodal distribution of aberrant cell frequencies was clearly detectable, showing the existence of DEB-sensitive subjects belonging to the second mode (CA frequencies > 19%). When responsiveness evaluated by means of CA induction was compared with SCE responsiveness, it was noted that all SCE-inducible subjects (> 110.9 SCEs/cell) belonged to the second mode of CA frequency distribution. On the other hand, highly CA inducible individuals did not necessarily show a higher SCE-response, although their DEB-induced SCE frequencies were above average (92 SCEs/cell). DEB-induced CA frequency correlated with baseline levels, indicating that DEB-sensitive individuals also showed higher spontaneous chromosome damage (3.6 versus DEB-resistant 2%, P < 0.05). Finally, when simple and multiple regression analyses were carried out, DEB-sensitivity appeared negatively related to haematic concentrations of proteins and uric acid (intercept 0.131 +/- 0.011, slope -0.029 +/- 0.0116, r = -0.39; P < 0.01), probably due to its antioxidant activity. This finding confirmed previous observations on the scavenger activity of plasma factors on DEB mutagenicity

The eps8 Family of Proteins Links Growth Factor Stimulation to Actin Reorganization Generating Functional Redundancy in the Ras/Rac Pathway

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice