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Fig. 7. FAK and Src phosphorylate UNC5 in vitro. (A) UNC5 phosphorylation by FAK and Src in vitro. Recombinant MBP-UNC5 purified from bacteria was used as a substrate for FAK and Src. FAK and Src were immuno-affinity purified from transfected HEK293 cells under stringent conditions. The presence of PP2 inhibitor in kinase reactions is indicated. In vitro kinase reaction was carried out by incubating UNC5 protein with FAK or Src in a solution contain [-³²P]ATP. Samples were analyzed by SDS-PAGE and autoradiography. Phosphorylation of UNC5, FAK and Src are indicated by arrows. The protein levels of MBP-UNC5 used in kinase reactions were detected by Coomassie staining. (B) Phosphorylation of UNC5 and mutants by Src. In vitro phosphorylation of UNC5 truncation mutants by Src were performed. UNC5 was purified from bacterial expression. Src was immunoprecipitated from transfected HEK293 cells. Phosphorylation of UNC5 and Src was detected by autoradiograph while MBP-UNC5 protein was detected by Coomassie staining. The diagram of UNC5 beneath Fig. 7B indicates the truncation mutants and corresponding tyrosine residues of UNC5 used in the experiment. DB, DCC-binding motif; DD, death domain; IG, immunoglubin domain; TM, transmembrane domain; TSP, thrombospondin domain; ZU-5, ZO-1/UNC5 domain. (C) The UNC5 M3 mutant does not induce cell shrinkage in Cos-7 cells. Cos-7 cells were transfected with wild-type UNC5, and M3 and C mutants. After 24 hours, cells were treated with AP-netrin. Cell morphology was observed by microscope. Bar, 50 µm.

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