Fig. 6. The RetDN mutant has decreased intrinsic kinase activity and inhibits wild-type Ret activity. (A) Western blots of extracts from CHP126 cells infected with the indicated Ret lentiviruses in the presence or absence of GDNF (25 ng/ml) were probed with the indicated antibodies. Wild-type Ret9 showed ligand-dependent autophosphorylation (pY20 and pY1062), and phosphorylation of downstream AKT and MAPK (pAKT and pMAPK, respectively), but RetDN and a kinase-inactive mutant with a K758M mutation (RetKD) lack these activities. (B) Immunoprecipitation studies demonstrated an interaction between RetDN and Ret9. Wild-type Ret9 (FLAG tagged) and RetDN (HA tagged) were co-expressed in 293T cells (which lack endogenous Ret). Lysates were immunoprecipitated with either FLAG- or HA-epitope specific antibodies, followed by western blotting (WB) using a pan-Ret antibody or a FLAG antibody to detect Ret9 and RetDN complexes. (C) RetDN inhibited ligand-dependent AKT phosphorylation. The human RET9 or RetDN were expressed in GDNF-responsive Neuro2A1 cells using lentivirus infection. The cells were grown in the presence or absence of GDNF, and western blots containing these cell lysates were probed with the indicated antibodies (pMAPK, phosphor-MAPK; pAKT, phosphor-AKT). Cells infected with RetDN, but not with wild-type Ret9, had markedly decreased levels of GDNF-dependent AKT phosphorylation, whereas MAPK phosphorylation was minimally affected. An antibody to AKT indicated equivalent total AKT levels in each sample. Control lanes represent lysates from cells infected with virus expressing only the Venus reporter. (D) Quantification of decreased AKT phosphorylation. The RetDN inhibition of AKT phosphorylation was quantified by normalizing the samples according to the total AKT levels and then comparing the GDNF-stimulated phospho-AKT levels. RetDN inhibited GDNF-mediated AKT phosphorylation by approximately 70% (n=3, ^*=P<0.01, mean±s.e.m.). (E) RetDN inhibited endogenous mouse wild-type Ret activity in SCG neurons. Immunoblots of extracts from primary SCG neurons (cultured for 8 days) show decreased GDNF-dependent phosphorylation of Ret Y1062, AKT and MAPK in Ret^DN/+ mice, when compared with that of Ret^DNneo/+ and wild-type mice (WT). The blot was re-probed with tubulin antibody to ensure equal loading.