Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1

Two human hSos1 isoforms (Isf I and Isf II; Rojas et al,, Oncogene 12, 2291 -2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representa tive NIH3T3 transfectants overexpressing either one. We showed that hSos1-I sf II is significantly more effective than hSos1-Isf I to induce proliferat ion or malignant transformation of rodent fibroblasts when transfected alon e or in conjunction with normal H-Ras (Gly12), The hSos1-Isf II-Ras cotrans fectants consistently exhibited higher saturation density, lower cell-doubl ing times, increased focus-forming activity and higher ability to grow on s emisolid medium and at low serum concentration than their hSos1-Isf I-Ras c ounterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21(ras) w as consistently higher in the hSos1-Isf II-transfected clones, both under b asal and stimulated conditions. However, no significant differences were de tected in vivo between Isf I- and Isf II-transfected clones regarding the a mount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation, Interestingly, di rect Ras guanine nucleotide exchange activity assays in cellular lysates sh owed that Isf II transfectants consistently exhibited about threefold highe r activity than Isf I transfectants under basal, unstimulated conditions. M icroinjection into Xenopus oocytes of purified peptides corresponding to th e C-terminal region of both isoforms (encompassing the 15 amino acid insert ion area and the first Grb2-binding motif) showed that only the Isf II pept ide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progeste rone, in induction of GVBD. Our results suggest that the increased biologic al potency displayed by hSos1-Isf II is due to higher intrinsic guanine nuc leotide exchange activity conferred upon this isoform by the 15 a.a. insert ion located in proximity to its Grb2 binding region.