The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1.
We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.
Nielsen
K H
KH
Department of Molecular Cell Biology, University of Copenhagen, Denmark.
Papageorge
A G
AG
Vass
W C
WC
Willumsen
B M
BM
Lowy
D R
DR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors; D066298:In Vitro Techniques; D051379:Mice; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014162:Transfection; D014452:Ubiquitins / Q000737:chemistry; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism*
D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins