Characterization of AND-34 Function and Signaling

AND-34 is a member of a novel family of proteins (NSP1, NSP2, and NSP3) that have an amino-terminal SH2 domain but bind by a carboxy-terminal GEF (Cdc25)-like domain to the carboxy-terminus of the focal adhesion adapter protein p130Cas. Direct GEF activity of AND-34 toward Ras subfamily members has not been demonstrated with purified protein. Overexpression of AND-34 in epithelial breast cancer cells leads to activation of Rac and Cdc42 by a PI3K-dependent mechanism. This chapter will describe the techniques we used to examine AND-34-induced Rac, Cdc42, Akt, and PAK1 activation in human breast cancer cell lines and in murine lymphoid cell lines. In addition, we summarize techniques used to determine that AND-34 overexpression does not activate R-Ras in MCF-7 cells.