Functional roles for the pleckstrin and Dbl homology regions in the Ras exchange factor Son-of-sevenless.
Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras.
McCollam
L
L
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
Bonfini
L
L
Karlovich
C A
CA
Conway
B R
BR
Kozma
L M
LM
Banerjee
U
U
Czech
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004330:Drosophila; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020664:Son of Sevenless Proteins; D013329:Structure-Activity Relationship
D001798:Blood Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D020664:Son of Sevenless Proteins; C039077:platelet protein P47; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)