|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. Functional importance of focal adhesion kinase (FAK) in UNC5 phosphorylation. (A) The P3 domain in DCC is essential for DCC to enhance UNC5 tyrosine phosphorylation. UNC5 was transfected with wild-type DCC, P1, P2 and P3 deletion mutants in HEK293 cells. Netrin stimulation for 20 minutes was indicated. Phosphorylation of immunoprecipitated UNC5 was determined as described in Fig. 1A and B. (B) The P3 domain of DCC is required for interaction with FAK. HA-FAK and different DCC deletion mutants were transfected into HEK293 cells. HA-FAK was immunoprecipitated and the co-precipitated DCC was detected by anti-DCC immunoblot. (C) DCC antibody blocks netrin-induced UNC5 tyrosine phosphorylation. DCC antibody directed against the extracellular domain in DCC was used to pretreat neurons isolated from dorsal spinal cord for 1 hour and then cells were treated with netrin for 20 minutes. Lysates from treated cells were immunoprecipitated with UNC5 antibody and the immunoprecipitates were used in a phosphotyrosine blot. (D) FAK stimulates basal and netrin-induced UNC5 tyrosine phosphorylation. UNC5 was transfected with or without DCC and FAK in HEK293 cells as indicated. 24 hours after transfection, cells were serum-starved for 8 hours before netrin stimulation. Protein levels of UNC5, DCC and FAK were detected by western blot with specific antibodies. Two exposures (short and long as indicated) of anti-phosphotyrosine immunoblots of UNC5 were shown. (E) FRNK, a dominant negative FAK, blocks UNC5 tyrosine phosphorylation in response to netrin. UNC5 was transfected with DCC and FRNK as indicated. The experiment was conducted under conditions similar to those for panel D. Lanes 5 and 6 are duplicated, except the amount of FRNK DNA used in lane 6 was increased. (F) FRNK inhibits phosphorylation of DCC and UNC5. Experiments are similar to those in panel E. Tyrosine phosphorylation of immunoprecipitated DCC and UNC5 were determined. Netrin treatment was 10 minutes on lane 3, the other lanes were treated by netrin for 20 minutes. Lane 6 and lane 7 are duplicated.
|
