Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291± 2300 de®ned by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is signi®cantly more eective than hSos1-Isf I to induce proliferation or malignant transformation of rodent ®broblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower celldoubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21 ras was consistently higher in the hSos1-Isf IItransfected clones, both under basal and stimulated conditions. However, no signi®cant dierences were detected in vivo between Isf I-and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of puri®ed peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the ®rst Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.

Purified, bacterially expressed PH domains of Sos1, IRS-1, ␤ARK, and PLC␦ 1 were analyzed functionally by means of microinjection into full grown, stage VI Xenopus laevis oocytes. Whereas the PH domains from IRS-1, ␤ARK, or PLC␦ 1 did not show any effect in the oocytes, injection of the purified Sos1 PH domain resulted in induction of significant rates of germinal vesicle breakdown and meiotic maturation. Furthermore, the Sos1 PH domain exhibited also significant synergy with insulin or coinjected normal Ras protein in induction of germinal vesicle breakdown, although it did not affect the rate of progesterone-induced maturation. These results suggest that purified, isolated PH domains retain, at least in part, their functional specificity and that Xenopus oocytes may constitute a useful biological system to analyze the functional role of the Sos1 PH domain in Ras signaling pathways. Pleckstrin homology (PH)1 domains are modular domains of about 100 amino acids present in a variety of signaling and cytoskeletal proteins, which were initially defined by their homology with the internal repeats of pleckstrin, a major protein kinase C substrate in platelets (1, 2). Included among the many PH-containing proteins described so far are several that participate in Ras signaling pathways. Despite having a rather limited primary sequence consensus identity, the PH domains from different proteins appear to share a common tertiary structure consisting of seven antiparallel ␤-sheets and a carboxyl-terminal amphiphilic ␣ helix (3-6).The functional role of PH domains remains unclear. Some reports describe a role of PH domains in protein-protein interactions such as Akt homooligomerization (7) and association with G␤␥ subunits (8 -10) or protein kinase C (11, 12), whereas others point to interactions with membrane lipids such as phosphatidylinositol 4,5-bisphosphate (13-17). An interesting hypothesis suggests that all those different interactions may represent alternative methods for directing the implicated proteins to membrane locations. Genetic evidence has confirmed the functional importance of PH domains since there is an established association between specific point mutations in the PH domain of Bruton tyrosine kinase and X-chromosome linked human agammaglobulinemia or mouse inmunodeficiency (18 -21). Regarding Ras signaling, recent reports point to the presence of positive regulatory elements in the region of the Ras guanine nucleotide exchanger Sos1 protein that encompasses its PH (and also the contiguous DH) domain (22).Xenopus oocytes provide a useful experimental model for the functional analysis of signaling molecules, which are able to induce their meiotic maturation, i.e. germinal vesicle breakdown (GVBD). Xenopus oocytes possess at least two independent pathways leading to GVBD. In one, progesterone leads to decreased adenylate cyclase activity, with a resulting drop in overall cAMP levels and protein kinase A-dependent phosphorylations. In the other, insulin or IGF-1 triggers a cascade of phosphorylations initi...