The isoform-specific stretch of hSos1 defines a new Grb2-binding domain.
hSos1 isoform II, defined by the presence of a 15 amino acid stretch in its carboxy-terminal region, exhibits higher Grb2 affinity than hSos1 isoform I. In this study, we investigated the cause for this difference and observed that, in addition to the four currently accepted Grb2-binding motifs, a number of additional, putative SH3-minimal binding sites (SH3-MBS) could be identified. The isoform II-specific 15 amino acid stretch contained one of them. Indeed, we demonstrated by site-directed mutagenesis that these SH3-MBS were responsible for the Grb2 interaction, and we found that C-terminal fragments of the two hSos1 isoforms (lacking the four cannonical Grb2-binding motifs, but containing the SH3-minimal binding sites) were able to bind Grb2, with the isoform II fragment showing higher Grb2 affinity than the corresponding isoform I fragment. Furthermore, we provide evidence that C-terminal truncated mutants of either hSos1 isoform, containing only the SH3-minimal binding sites, were able to originate in vivo stable complexes with Grb2. Although, Grb2-binding remains higher in both full-length isoforms, compared to the C-terminal truncated mutants, these mutants were also able to activate Ras, supporting a potential role of this C-terminal region as negative modulator of Sos1 activity. These findings document the existence of a new, functional, SH3-minimal binding site located in the specific stretch of hSos1 isoform II which may be responsible for the increased Grb2 affinity of this isoform in comparison to isoform I, and for the physiological properties differences between both isoforms. Moreover, these SH3-minimal binding sites may be sufficient to attain stable and functional hSosl-Grb2 complexes.
Zarich
N
N
Unidad de Biología Celular, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Madrid, Spain.
Oliva
J L
JL
Jorge
R
R
Santos
E
E
Rojas
J M
JM
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020837:SOS1 Protein / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D010446:Peptide Fragments; D020033:Protein Isoforms; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020837:SOS1 Protein; D005982:Glutathione Transferase