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 system s. One of the functions of Grb2 is to link tyrosine-phosphorylated rec eptors to downstream effector proteins via the SH2 and SH3 domain bind ings. To identify Grb2-associated proteins in Ras transformed NIH3T3 c ells, we performed coprecipitation experiments using recombinant GST-G rb2 fusion proteins and found a remarkably strong band of 100 kDa. Wit h N-terminal amino acid sequencing, we identified the protein of 100 k Da as dynamin II. Dynamin II was also observed in the coprecipitates w ith 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 dy namin II was confirmed by competitive binding experiments with oligope ptides whose sequence corresponded to that of SH2 or SH3 binding motif . The dynamin II coprecipitation was completely abrogated by the addit ion of the oligopeptide of SH3 binding motif, but addition of SH2 bind ing motif had no effect. In conclusion, these results suggest that dyn amin II may be largely expressed and closely associated with Grb2-medi ated signaling in Ras transformed cells. (C) 1997 Academic Press.