Lnk SH2 mutants dominant-negatively inhibit the negative regulatory function of Lnk in cell growth. (A) Lnk associates with c-Kit via the SH2 domain. COS7 transfectants coexpressing the indicated Lnk mutants and c-Kit were stimulated with SCF and lysed. Proteins immunoprecipitated with an anti-Lnk antibody were separated and immunoblotted with an antiphosphotyrosine antibody to detect c-Kit (top panel) or with an anti-Lnk antibody (bottom panel). The association of c-Kit with Lnk was abolished by the R364E SH2 mutation. (B) Multimer formation of Lnk via the N-terminal region. Total cell lysates of COS7 transfectants expressing the indicated Lnk mutants were treated with various concentrations of the chemical cross-linker BS³  and then subjected to immunoblotting using anti-Lnk antibodies. Lnk formed multimer complexes with slower mobility (arrowhead). Multimerized complexes were not detected with the dN Lnk mutants. (C) Wild-type Lnk or dPH/RE/dC mutant associated and coimmunoprecipitated with Lnk-GFP by anti-GFP (arrowheads in bottom panel). In contrast, dN/dPH mutant did not coimmunoprecipitate with Lnk-GFP. (D-E) Lnk SH2 mutants were retrovirally transduced into MC9-Lnk cells expressing wild-type Lnk. The transduced cells were cultured in the presence of SCF, and their growth was compared with that of nontransduced MC9-Lnk cells by dividing the percentage of eGFP-positive cells at the indicated time points by that at the start of culture (day 0). Data shown are means ± SD of 3 experiments.