Signal transduction in T lymphocytes using a conditional allele of Sos.
While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.
Holsinger
L J
LJ
Howard Hughes Medical Institute, Stanford University School of Medicine, CA 94305, USA.
Spencer
D M
DM
Austin
D J
DJ
Schreiber
S L
SL
Crabtree
G R
GR
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000235:genetics*; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C084260:FK 1012; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins; D016559:Tacrolimus