Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSos1.
hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.
Zarich
Natasha
N
Unidad de Biología Celular, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
Oliva
José Luis
JL
Martínez
Natalia
N
Jorge
Rocío
R
Ballester
Alicia
A
Gutiérrez-Eisman
Silvia
S
García-Vargas
Susana
S
Rojas
José M
JM
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015536:Down-Regulation / Q000235:genetics*; D051380:GRB2 Adaptor Protein / Q000172:deficiency / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D020837:SOS1 Protein / Q000737:chemistry / Q000378:metabolism*
D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D050505:Mutant Proteins; D020837:SOS1 Protein