Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is a n essential element of the Ras signaling pathway in multiple systems. The S H2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several prolin e-rich domain-containing proteins such as Sos1. To define the difference in Grb2-associated proteins in hepatocircinoma cells, we performed coprecipit ation analysis using recombinant GST-Grb2 fusion proteins and found that se veral protein components (p170, p125, p100, and p80) differently associated with GST-Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cell s. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang l iver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST- Grb2 SH2 proteins exclusively bound to the p46(Shc), p52(Shc), and p66(Shc) are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N-SH3 and C-SH3 do mains of Grb2 fusion proteins coprecipitated with dynamin II besides Sosl. These results suggest that dynamin II may be a functional molecule involved in Grb2-mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. (C) 2001 Wiley-Liss, Inc.