Dynamin II associates with Grb2 SH3 domain in Ras transformed NIH3T3 cells.
Grb2, a linker protein containing two SH3 domains and one SH2 domain, is known as an essential element of the Ras pathway in multiple systems. One of the functions of Grb2 is to link tyrosine-phosphorylated receptors to downstream effector proteins via the SH2 and SH3 domain bindings. To identify Grb2-associated proteins in Ras transformed NIH3T3 cells, we performed coprecipitation experiments using recombinant GST-Grb2 fusion proteins and found a remarkably strong band of 100 kDa. With N-terminal amino acid sequencing, we identified the protein of 100 kDa as dynamin II. Dynamin II was also observed in the coprecipitates with the GST fusion protein of N-SH3 or C-SH3 domain of Grb2 but not in that of Grb2 SH2 domain. The SH3-mediated association of Grb2 with dynamin II was confirmed by competitive binding experiments with oligopeptides whose sequence corresponded to that of SH2 or SH3 binding motif. The dynamin II coprecipitation was completely abrogated by the addition of the oligopeptide of SH3 binding motif, but addition of SH2 binding motif had no effect. In conclusion, these results suggest that dynamin II may be largely expressed and closely associated with Grb2-mediated signaling in Ras transformed cells.
Yoon
S Y
SY
Immune Regulation RU, Korea Research Institute of Bioscience and Biotechnology, Yusung, Taejon.
Koh
W S
WS
Lee
M K
MK
Park
Y M
YM
Han
M Y
MY
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D034281:Dynamins; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D011905:Genes, ras; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D011506:Proteins; D020558:GTP Phosphohydrolases; D034281:Dynamins