a, CHO cells expressing EGFP–talin, or EGFP–tal_S425A were plated on 5 µg/ml fibronectin and analysed by time-lapse TIRF microscopy. Arrow heads points to lamellipodium formation and retraction. b, Kymograph of lamellipodium dynamics in CHO cells expressing EGFP–talin or EGFP–tal_S425A. c, The lamellipodial persistence in cells expressing wild-type Talin is significantly higher than that of mutant S425A. d, There is no significant difference in protrusion velocities between cells expressing the wild-type and mutant S425A. Quantifications are expressed as means ± SEM.

a, CHO cells stably expressing EGFP–talin, EGFP–tal_S425A, or EGFP-tal_S425D were plated on fibronectin (5µg/ml)-coated glass-bottom dishes (MatTek) and analyzed by time-lapse TIRF microscopy. Arrowheads point to disassembling focal adhesions. b, Quantification of the rate constants of EGFP–talin, EGFP–tal_S425A, or EGFP-tal_S425D assembly into and dissociation from focal adhesions. (c) Effects of roscovitine on the rate constants of EGFP–talin, assembly into and dissociation from focal adhesions. Quantifications are expressed as means ± SEM of 30 focal adhesions from 7 cells.

a, Inhibition of PDGF-stimulated migration of SH-SY5Y neuroblastoma cells by expressing EGFP-tal_S425A or –tal_S425D. SH-SY5Y cells were transfected with EGFP-talin, -talS425A or –talS425D, using Nucleofactor Kit V (Amaxa). At 24 hr post-transfection, cells were trypsinized for transwell migration assays employing collagen I (10µg/ml)-coated filters and 20 nM PDGF in lower charmber. c and d, Inhibition of wound closure in CHO cells expressing EGFP-tal_S425A or –tal_S425D. Confluent monolayers of CHO cells stably expressing EGFP-talin, tal_S245A or –tal_S425D were wounded and images were taken when the wounds were made (left panels) and after incubating for 18 hr (right panels). Data are expressed as mean ± SEM of three independent experiments.

a, Direct phosphorylation of TH by Cdk5 in vitro. Recombinant His-tagged TH (2.8 µg) was incubated with active Cdk5/p25 in 40 µl of kinase buffer containing 40 µM [γ-³²P]ATP (10 µCi). A 1:8 stoichiometry of phosphorylation of the TH was achieved in the experiments. b, Phosphorylation of Ser₄₂₅ on talin by Cdk5 in vitro. HA-talin, -tal_S425A or -tal_S425D were immunoprecipitated from transfected CHO-K1 cells, and incubated with active Cdk5/p35 in 50 µl of kinase buffer containing 20µM [γ-³²P]ATP (10 µCi). The numbers on the top indicates phosphorylation levels (% of wt talin) quantified as described in “Methods”. c, 2-D phospho-peptide mapping analysis of wt talin, tal_S425A, and tal_S425D. d, Talin is phosphorylated at Ser₄₂₅ by Cdk5 in vivo. Cells were transfected with HA-talin or –talS425A in the presence or absence of Cdk5/p35. After 18 hr of transfection, cells were incubated with [³²P]orthophosphoric acid (0.8 mCi/ml) for 12 hr. Cells were then harvested and analyzed as described in “Methods”. Over-expression of Cdk5 promotes phosphorylation of talin but not of tal_S425A. e, 2-D phospho-peptide mapping analysis of talin and tal_S425Ain the absence and presence of recombinant Cdk5.

a, Talin-Smurf1 interaction. Binding of EGFP-Smurf1 to immobilized GST-profilin, -tal1-433, -tal393-605, or -tal393-605_S425D. Smurf1 was detected by immunoblotting with anti-GFP. b, TH but not full-length talin binds Smurf1 tightly,. Binding of purified His-tagged TH or FL talin to immobilized GST-Smurf1 was detected by immunoblotting with anti-His tag mAb. c, TH and catalytically-inactive Smurf1(Sm_C699A) interact in cells. EGFP-TH and VSVG-Sm_C699A were co-transfected in combination with H-Ras_G12V into CHO-K1 cells. Anti-GFP immunoprecipitated EGFP-TH and bound Smurf1_C699A was detected with anti-VSVG. d, Inhibition of TH-Smurf1 interaction by Cdk5-mediated phosphorylation. HA-TH was purified from CHO-K1 cell lysates and phosphorylated in vitro with Cdk5/p35 in vitro, and binding to GST-Smurf1 was analyzed as in panel b. e, Inhibition of Smurf1-mediated ubiquitination Cdk5-mediated phosphorylation. Purified His-tagged TH was incubated 120 min with the indicated quantity of Cdk5/p25 and then with Myc-ubiquitin, E1, and UbcH7 in the presence or absence of GST-Smurf1. f, Effect of TH phosphorylation on ubiquitination in vivo. CHO-K1 cells were co-transfected with EGFP-TH, -TH_S452A, or -TH_S425D and HA-ubiquitini. Twenty-four hr post-transfection, cells were re-plated on fibronection for 5 hr and then were treated with MG132 (40µM) for an additional 4 hr before ati-HA immunoblotting. g, The poly-ubiquitination of EGFP-TH, -TH_S452A and -TH_S425D was quantified by densitometry of the rectangular area (see lane 3). Depicted are the mean and range of two independent experiments. h, CHO cells expressing EGFP–tal_S425A, were transfected with vector or DsRed-Smurf1_C699A and plated on fibronectin for 4 hr. The rate constants of EGFP–tal_S425A dissociation from focal adhesions were quantified by time-lapse microscopy (mean ± SEM, n=30). i, Proposed mechanism whereby talin phosphorylation by Cdk5 regulates TH ubiquitination, focal adhesion disassembly, lamellipodial dynamics, and cell migration. Full scans of western blots shown in b, c, e, and f are available in Supplementary Information, Fig. S7.