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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02697

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FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5

¹ Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
² Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
³ Department of Neurobiology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA
⁴ Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109, USA

View larger version (81K): [in a new window]   Fig. 1. Netrin and DCC induce UNC5 tyrosine phosphorylation. (A) Netrin-induced tyrosine phosphorylation of UNC5 in spinal cord neurons. Spinal cords were dissected from E15 mouse embryos. Neuron cultures were starved for at least 4 hours in serum-free DMEM medium. Netrin (200 ng/ml) was added to the indicated well for 5, 10 and 20 minutes. Lane 2 was pretreated with PP2. Neurons were lysed in PLC buffer and immunoprecipitated with anti-UNC5 antibody. Phosphotyrosine antibody was used to detect phosphorylated UNC5 protein. IB, immunoblot; IP, immunoprecipitation; pY, anti-phosphotyrosine blot. (B) DCC is required for netrin to induce UNC5 tyrosine phosphorylation. UNC5 was transfected with or without DCC in HEK293 cells as indicated. Transfected cells were serum-starved for 8 hours before stimulation. Netrin was added to the indicated lane for 5, 10 and 20 minutes and cells were lysed in PLC buffer. Tyrosine phosphorylation of immunoprecipitated UNC5 was detected by anti-phosphotyrosine immunoblot. (C) The intracellular domain of UNC5 is required for morphological changes in COS-7 cells. COS-7 cells were transfected with DCC wild-type, UNC5 wild-type and their intracellular domain deletion mutants as indicated. After a 24 hour transfection, transfected cells were treated with AP-netrin for 30 minutes. Alkaline phosphatase stained for AP-netrin, which bound to UNC5- and/or DCC-expressing cells (dark color). Bar, 100 µm.

View larger version (40K): [in a new window]   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.

View larger version (27K): [in a new window]   Fig. 3. FAK is required for netrin-induced UNC5 tyrosine phosphorylation. (A) Netrin fails to stimulate UNC5 tyrosine phosphorylation in FAK-/- MEF. UNC5 was transfected with DCC into wild-type and FAK-knockout MEF cells. 36 hours after transfection, cells were serum-starved for about 8 hours followed by netrin stimulation (200 ng/ml) for 20 minutes. UNC5 protein was immunoprecipitated by anti-HA antibody. Immunoprecipitates were subjected to anti-phosphotyrosine immunoblot. (B) Expression of FAK restores the ability of netrin to stimulate UNC5 tyrosine phosphorylation. FAK (10 ng) was co-transfected with UNC5 and DCC into FAK-/- MEF cells. The experiment was conducted under similar conditions to those described in panel A.

View larger version (41K): [in a new window]   Fig. 4. Src family kinases are important for UNC5 tyrosine phosphorylation in response to netrin. (A) PP2 inhibits netrin-stimulated UNC5 tyrosine phosphorylation. UNC5 and DCC were transfected into HEK293 cells. Before netrin treatment, PP2, a Src family kinase inhibitor, was added to the culture for 30 minutes. The phosphotyrosine immunoblot showed that PP2 inhibited netrin-induced UNC5 tyrosine phosphorylation. (B) Src enhances UNC5 phosphorylation. UNC5 was transfected with wild-type Src kinase (lanes 2 and 3) and Src (K/M), a kinase-dead mutant (lane 4). Lane 3 was treated with PP2. Anti-HA immunoprecipitates were subjected to immunoblotting with phospho-tyrosine antibody.

View larger version (36K): [in a new window]   Fig. 5. Src family kinases are required for netrin to induce UNC5 tyrosine phosphorylation. (A) Netrin fails to induce UNC5 tyrosine phosphorylation in Src/Fyn/Yes triple-knockout MEF cells. UNC5 was transfected with DCC into wild-type and Src/Fyn/Yes triple-knockout MEF cells. 48 hours after transfection, cells were serum-starved for 8 hours and netrin was added for the indicated times. Tyrosine phosphorylation of immunoprecipitated UNC5 was determined. (B) Expression of Src restores netrin-induced UNC5 tyrosine phosphorylation in Src/Fyn/Yes triple-knockout cells. UNC5 protein from transfected cells was immunoprecipitated and subjected to immunoblot with anti-phosphotyrosine antibody. Transfections of Src, UNC5 and DCC are indicated.

View larger version (41K): [in a new window]   Fig. 6. UNC5 associates with Src. (A) Co-immunoprecipitation of Src and UNC5 in transfected HEK293 cells. UNC5 was transfected with wild-type Src or the kinase-dead Src mutant into HEK293 cells. 48 hours after transfection, transfected cells were lysed and immunoprecipitated by anti-Src antibody. Immunoprecipitates were subjected to anti-HA immunoblot. (B) The SH2 domain in Src is required for the interaction with UNC5. UNC5 was transfected with wild-type Src, SH3 or SH2 Src mutants into HEK293 cells. Coimmunoprecipitation was conducted as described for panel A. Anti-HA immunoblot showed that the Src SH2 mutant significantly decreased the interaction with UNC5. (C) Co-immunoprecipitation of endogenous UNC5 by anti-Src antibody. Brain tissues from E15 mouse embryos were dissected and lysed in NP40 buffer and were used for immunoprecipitation by anti-Src antibody. Immunoprecipitates were subjected to anti-UNC5 immunoblot. Lysate was used as a positive control. (D) Co-immunoprecipitation of endogenous Src by anti-UNC5 antibody. Experiments were similar to panel C. (E) Mapping the region in UNC5 responsible for the interaction with Src. UNC5 recombinant proteins purified from bacteria expressing deletion mutants of UNC5 were used in an in vitro pull-down experiment to test their interaction with Src. Src was immunoprecipitated from transfected HEK293 cells. Src binding on beads was equally divided into each reaction and incubated with different MBP-UNC5 mutants. The UNC5 protein associated with Src was determined by HA immunoblot. UNC5 protein input was shown by Coomassie staining (middle panel).

View larger version (47K): [in a new window]   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.

View larger version (20K): [in a new window]   Fig. 8. A model showing the involvement of FAK and Src in UNC5-DCC signaling. FAK interacts via its CTD with the P3 domain of DCC. Src interacts via its SH3 domain with the P1400XXP motif in DCC and via its SH2 domain with phosphorylated UNC5 or FAK. FAK and Src phosphorylate Y1420 in DCC and multiple tyrosine sites in UNC5. Note that molecules are not drawn to scale. CTD, C-terminal domain; DB, DCC-binding motif; DD, death domain; FN, fibronectin type III domain; IG, immunoglobulin domain; NTD, N-terminal domain of FAK; P1, P2 and P3, three conserved domains; TSP, thrombospondin domain; ZU-5, ZO-1/UNC5 domain. The numbers 449/454 and 649/667 indicate sites in UNC5 phosphorylated by Src.