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

Abstract

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.

hSos1 Contains a New Amino-terminal Regulatory Motif with Specific Binding Affinity for Its Pleckstrin Homology Domain

“…In support of this idea, constitutive or conditional membrane targeting of these exchange factors has been shown to strengthen Ras activation in transfected cells (11,12). However, some reports suggest that regardless of sub-cellular location, the intrinsic Ras guanine nucleotide exchange activity of Sos (Ras-GEF activity) may differ before and after stimulation of surface tyrosine kinase receptors (13,14).…”

“…DNA Constructs-The plasmids pCEFL-KZ-HA, pCEFL-KZ-AU5, pCEFL-KZ-HA-hSos1, pCEFL-KZ-HA-NDP, pCEFL-KZ-AU5-H-Ras wt, pCEFL-KZ-AU5-H-RasV12, pGEX-4T-1-NDP, pGal4-Luc, and pCDNAIII-Gal4-Elk-1 were described previously (14,19,21). The HF motif, DH, PH, and DH-PH domains of hSos1 (coding regions 1-640, 537-1326, 1263-1701, and 537-1653, respectively) were PCR-amplified from pCEFL-KZ-HA-hSos1 using the specific primers and providing sites BglII and NotI at the 5Ј and 3Ј ends, respectively.…”

Exchange Factors of the RasGRP Family Mediate Ras Activation in the Golgi

“…4, fifth and sixth panels from top). This may be because of the higher Grb2 binding affinity of the Sos1.1 isoform (11). No re-localization of EGFP-RBD was detected toward the Golgi apparatus in the presence of either RasGRF2 or Sos1 proteins (Fig.…”

“…To this end, we transfected COS1 cells with mammalian expression vectors encoding epitope-tagged versions of two Sos1 isoforms, of RasGRF2, and of three RasGRP family members (RasGRP1, -2, and -3). The two Sos1 proteins (isoforms 1 and 2) differ on the presence of a 15-amino acid stretch located adjacent to the first proline-rich region required for Grb2 binding (11). After transfection with those plasmids, cells were fixed and subjected to immunofluorescence analysis with the appropriate combination of antibodies.…”

The cyclopentenone 15-deoxy-Δ 12,14 -prostaglandin J 2 binds to and activates H-Ras

“…The plasmids used have been described (26)(27)(28)(29). The mutants C118S and C184S of H-Ras were obtained by PCR from pCEFL-KZ-AU5-H-Ras WT, using specific primers providing BglII and NotI sites at the 5Ј and 3Ј ends, respectively.…”