Supplemental Data Supplementary Figure Legends and Scheme Figure S1. UTK1 inhibits the second EGF-induced wave of lamellipodia formation in TT cells. A and B, EGF-induced lamellipodia formation in TT cells, (A) observed under confocal microscopy, (B) and counted. C-F, effect of UTK1 on EGF-induced lamellipodia formation. TT cells were pretreated with the indicated concentrations of UTK1 for 15 min and stimulated with EGF. After 10 min (C and D) or 12 h (E and F), the cells were observed under confocal microscopy (C and E), and counted (D and F). Data represent means SD (n = 6). G, inhibitory activity of UTK1 on EGF-induced cell migration, monitored using a chemotaxis chamber. Data represent means SD (n = 5). Arrows, lamellipodia. Scale bar, 10!m. For F, statistical analyses were performed with a two-tailed Student s t-test; *, P = 0.0019; **, P = 4.7 x 10-5. Throughout, the data was representative of at least three independent studies. For B, D and F, more than 300 cells were analyzed per experiment. Figure S2. UTK1 inhibits the second EGF-induced wave of Rac1 activation in TT cells. A, time-course analysis of Rac1 activation following EGF stimulation. TT cells were stimulated with EGF for the indicated periods, then the cells were examined for active Rac1 by pull-down assay. B and C, effect of UTK1 on EGF-induced Rac1 activation. TT cells were pretreated with UTK1 for 12 h (B) or 15 min (C), and stimulated with EGF. Following 10 min (B) or 9 h (C) of incubation, the cells were examined for active Rac1 by pull-down assay. Throughout, the data was representative of at least three independent studies. Figure S3. B-UTK1ox binds to the C-terminal region of 14-3-3!. A, schematic illustration of wild type and mutants of GST-14-3-3! for in vitro B-UTK1 pull-down assay. B, in vitro B-UTK1 pull-down assay. Purified GST-14-3-3! proteins (GST only, C50, "C200, "C100, or WT) were incubated with B-UTK1ox and avidin beads. The precipitated proteins were subjected to western blotting using anti-gst antibody. Throughout, the data was representative of at least three independent studies. Figure S4. Knockdown of both Tiam1 and #Pix does not affect the first EGF-induced wave of Rac1 activation. A, control, Tiam1, or #Pix sirna-transfected A431 cells were stimulated with EGF for 2 min. Then, the cells were examined for active Rac1 by pull-down assay. B, control, Tiam1, or
#Pix sirna-transfected A431 cells were stimulated with EGF for 5 min. Then, the cells with lamellipodia were counted. Data represent means SD (n = 6). Throughout, the data was representative of at least three independent studies. For B, more than 300 cells were analyzed per experiment. Figure S5. 14-3-3! and Tiam1 are involved in the second EGF-induced wave of Rac1 activation in TT cells. A, TT cells were transfected with control, Tiam1, #Pix, or 14-3-3! sirna and were cultured for 72 h. Then the cells were subjected to western blotting using the indicated antibodies. B, control, Tiam1, #Pix, or 14-3-3! sirna-transfected TT cells were incubated in the upper chamber and stimulated with or without EGF for 24 h. Then, the migrated cells were counted. Data represent means SD (n = 5). C, control, Tiam1, #Pix, or 14-3-3! sirna-transfected TT cells were stimulated with EGF for 9 h. Then, the cells were examined for active Rac1 by pull-down assay. For B, statistical analyses were performed with a two-tailed Student s t-test; *, P = 4.1 x 10-7 ; **, P = 4.3 x 10-8. Throughout, the data was representative of at least three independent studies. Figure S6. Time-course analysis of Tiam1 expression in EGF-treated A431 cells. A431 cells were treated with EGF for the indicated periods. The cells were lysed and subjected to western blotting using the indicated antibodies. The data was representative of at least three independent studies. Figure S7. NSC23766 inhibits the second EGF-induced wave of Rac1 activation but not the first wave. A431 cells were pretreated with NSC23766 for 12 h (A) and 15 min (B) and stimulated with EGF. Following 2 min (A) or 12 h (B) of incubation, the cells were examined for active Rac1 by pull-down assay. C, Effect of NSC23766 on EGF-induced cell migration in A431 cells, monitored using a chemotaxis chamber. Data represent means SD (n = 5). Throughout, the data was representative of at least three independent studies. Figure S8. Effect of UTK1 on expression levels and intracellular localization of Tiam1 protein. A, A431 cells were pretreated with UTK1 for 15 min and stimulated with EGF. Following 12 h of incubation, the cells were lysed and subjected to western blotting using the indicated antibodies. B, A431 cells were co-transfected with Tiam1-6 x myc and FLAG-14-3-3!. The cells were stimulated with EGF for 12 h in the absence or presence of 3!M UTK1. Then the cells were fixed and immunostained, and observed under confocal microscopy. Scale bar, 10!m. Throughout, the data was representative of at least three independent studies.
Figure S9. Asef and Vav2 do not interact with 14-3-3!. Lysates of A431 cells stimulated with EGF for 2 min or 12 h were incubated with GST or GST-14-3-3!. The binding proteins of GST or GST-14-3-3! precipitated with Glutathione Sepharose 4B were subjected to western blotting using the indicated antibodies. The data was representative of at least three independent studies. Scheme 1. Preparation of biotinylated compounds of UTK1 Biotinylated UTK1s (B-UTK1ph (3) and B-UTK1ox (6)) were synthesized as shown in Scheme 1. UTK1 (1), prepared according to our procedure 1, was directly converted to B-UTK1ph (3) by treatment with 2. B-UTK1ox (6) was synthesized via 5 by oxmation, reduction of the azide and coupling with 2.
A Fig. S1 0 h 10 min 90 min 6 h 10 h 12 h 14 h 16 h B 100 Lamellipodia formation (%) 50 0 0 2 4 6 8 10 12 14 16 Time (h)
Fig. S1 (continued) C Lamellipodia formation at 10 min UTK1 0 0 1 3!M EGF D 80 Lamellipodia formation (%) 40 0 0 0 0.3 1 3 UTK1 (!M) EGF 1 ng/ml
Fig. S1 (continued) E Lamellipodia formation at 12 h UTK1 0 0 1 3!M EGF F 30 * * * Lamellipodia formation (%) 20 10 0 0 0 0.3 1 3 UTK1 (!M) EGF 1 ng/ml
Fig. S1 (continued) G 80 Cells / field 40 0 0 0 0.3 1 3 UTK1 (!M) EGF 1 ng/ml
Fig. S2 A EGF 1 ng/ml 0 10 60 180 360 540 720 B 1st wave of Rac1 activation (at 10 min) in TT cells UTK1 0 0 0.3 1 3!M EGF 1 ng/ml Rac1-GTP Rac1 C 2nd wave of Rac1 activation (at 9 h) in TT cells UTK1 0 0 0.3 1 3!M EGF 1 ng/ml Rac1-GTP Rac1
Fig. S3 A WT GST 1 45 46 145 146 195 196 binding to UTK1 245 a.a. o!c100 GST x!c200 GST x C50 GST o GST GST x B GST C50!C200!C100 WT Pull-down: B-UTK1ox anti-gst Input anti-gst
Fig. S4 A 1st wave of Rac1 activation (at 2 min) Control Tiam1 "Pix sirna EGF 30 ng/ml Rac1-GTP Rac1 B 1st wave of lamellipodia formation (at 5 min) None EGF 30 ng/ml 100 Lamellipodia formation (%) 75 50 25 0 Control Tiam1!Pix sirna
Fig. S5 A Control Tiam1 "Pix 14-3-3# sirna Tiam1 "Pix 14-3-3# "-actin B Cell migration None EGF 1 ng/ml * * * 80 Cells / field 40 0 Control Tiam1!Pix 14-3-3" sirna
Fig. S5 (continued) C 2nd wave of Rac1 activation (at 9 h) in TT cells Control Tiam1 "Pix sirna EGF 1 ng/ml Rac1-GTP Rac1 Control 14-3-3# sirna EGF 1 ng/ml Rac1-GTP Rac1
Fig. S6 EGF 30 ng/ml 0 2 30 180 360 540 720 min Tiam1 "-actin
Fig. S7 A NSC23766 0 0 10 30!M EGF 30 ng/ml (2 min) Rac1-GTP Rac1 B NSC23766 0 0 10 30!M EGF 30 ng/ml (12 h) Rac1-GTP Rac1 C 120 cells / field 80 40 0 0 0 10 30 NSC23766 (!M) EGF 30 ng/ml
Fig. S8 A UTK1 0 0 0.3 1 3!M EGF 30 ng/ml Tiam1 "-actin
Fig. S8 (continued) B EGF EGF + UTK1 Merge F-actin FLAG-14-3-3# Tiam1-6 x myc
Fig. S9 EGF stimulation 2 min lysate Input GST GST- 14-3-3# EGF stimulation 12 h lysate Input GST GST- 14-3-3# Pull-down Asef Vav2
Scheme 1 cis UTK1 (1) C + N N N 5 5 2 S N N K 2 C 3, DMF rt, 2 d., 19% cis C 5 N 5 N B-UTK1ph (3) S N N UTK1 (1) + N 3 N 2 4 (ref.2 MS 4A, DCM rt,.n., 47% cis N 5 N 3 PMe 3, TF/ 2 = 10:1, rt, 1 h cis then 2,DMF rt,.n., 58% B-UTK1ox (6) N N 5 N 5 N S N N
Supplementary Methods Materials EGF was purchased from Sigma (St. Louis, M). NSC23766 was purchased from Calbiochem (San Diego, CA). Mouse monoclonal anti-rac1 (clone 23A8) and mouse monoclonal anti-kinesin (clone 2) antibodies were purchased from Millipore (Bedford, MA). Mouse monoclonal anti-gst (B-14), rabbit polyclonal anti-14-3-3! (C-16), rabbit polyclonal anti-14-3-3$ (C-17), rabbit polyclonal anti-tiam1 (C-16), and mouse monoclonal anti-myc (9E10) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-#-actin (AC-74), and mouse monoclonal anti-flag (M2) antibodies were purchased from Sigma. Mouse monoclonal anti-14-3-3% antibody was purchased from BD Transduction Laboratories (San Diego, CA). Rabbit polyclonal anti-flag (used for immunostaining) and rabbit polyclonal anti-#pix antibodies were purchased from Cell Signaling Technology (Beverly, MA). orseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from GE ealthcare (Little Chalfont, UK). Cell culture uman epidermal carcinoma A431 cells and A431 cells stably expressing FLAG-14-3-3! (A431/FLAG-14-3-3! cells) were maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 5% calf serum (CS). uman esophageal carcinoma TT cells were maintained in RPMI1640 (Nissui) supplemented with 5% fetal bovine serum (FBS). Prior to stimulation with EGF, A431 cells and A431/FLAG-14-3-3! cells were serum-starved by incubation in DMEM supplemented with 0.2% CS, and TT cells were serum-starved by incubation in RPMI1640 supplemented with 1% FBS for 15 min, respectively. Chemotaxis chamber assay using TT cells TT cells suspended in RPMI supplemented with 1% FBS were incubated in the upper chamber; the lower chamber contained RPMI supplemented with 1% FBS in the presence or absence of EGF (1 ng/ml). Drugs were added to both chambers. Following 24 h incubation, the filter was fixed with Me and stained with hematoxylin. The cells attached to the lower side of the filter were counted. Establishment of A431 cells stably expressing FLAG-14-3-3! A431 cells were transfected with pcdna3/flag-14-3-3! using Metafectene Pro (Biontex). Transfected cells were selected by 400!g/mL G418. Stable expression of FLAG-14-3-3! was verified by western blotting.
Western blotting Cells were lysed with IP buffer containing 1% NP-40 and 0.1% SDS. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore) by electroblotting. After the membranes had been incubated with primary and secondary antibodies, the immune complexes were detected with an Immobilon Western kit (Millipore), and the luminescence was detected with a LAS-1000 mini (Fujifilm, Tokyo, Japan). Co-transfection and immunostaining A431 cells were transiently co-transfected with 0.5!g of pcdna3/flag-14-3-3! and 0.5!g of pcs2+mt/tiam1 using Neon transfection system (Invitrogen). The cells were stimulated with EGF for 12 h in the absence or presence of UTK1 (3!M). Then the cells were fixed, permeabilized, and incubated in blocking buffer (1% bovine serum albumin in PBS) for 30 min. The cells were incubated with anti-flag antibody (1:200, Cell Signaling) and anti-myc antibody (1:500, Santa Cruz) in blocking buffer at 4 C for overnight. After rinsing three times with TBS-Tween (20 mm Tris-Cl (p 7.6), 137 mm NaCl, and 0.1% Tween 20) and once with blocking buffer, the cells were incubated with anti-rabbit-alexa 488 (1:1000, Molecular Probes) and anti-mouse-alexa 647 (1:1000, Molecular Probes) at room temperature for 1 h followed by staining with Texas Red phalloidin. Fluorescence images were obtained using a confocal laser scanning microscope system FV1000. RNA interference sirna for control (12935-300), 14-3-3! (SS111443), Tiam1 (SS110756), and #Pix (SS113109) were purchased from Invitrogen. A431 and TT cells were transfected with sirna using iperfect (QIAGEN, ilden, Germany) according to the manufacturer s instructions. Characterization of UTK1, B-UTK1ph, and B-UTK1ox Spectral data of UTK1 (1 in Scheme 1): 1 NMR (300 Mz CDCl 3 )! (ppm) 0.78-1.52 (9, m), 0.85 (1.5, d, J = 3.0 z), 0.88 (1.5, d, J = 3.0 z), 0.89 (3, s), 0.95 (3, s), 0.96 (3, s), 1.92 (1, m), 2.09 (2, m), 2.49 (3, s), 2.79 (0.5, dd, J = 7.5 z, 15.0 z), 2.80 (0.5, dd, J = 7.5 z, 15.0 z), 3.10 (1, dd, J = 1.8 z, 15.0 z), 4.41 (1, d, J = 7.5 z), 4.90 (1, s), 5.04 (1, s), 6.31 (1, s), 9.21 (1, br), 10.03 (1, s), 12.77 (1, s). 13 C NMR (125 Mz, CDCl 3 )! (ppm) 17.89, 19.02, 19.14, 21.80, 21.95, 24.53, 28.31, 28.49, 28.84, 28.98, 29.27, 30.87, 30.99, 31.11, 34.71, 34.76, 34.95, 34.99, 36.51, 36.68, 50.17, 50.23, 77.11, 77.15, 108.85, 108.88, 111.27, 112.71, 113.61, 143.13,
151.94, 165.10, 165.39, 193.23. IR (KBr) & = 3676-3153 (br), 2923, 2866, 1624, 1484, 1447, 1385, 1368, 1276, 1249, 1197 cm -1. ESI-RMS m/z calcd for C 23 34 Na 4 [M+Na] + 397.2349, found 397.2323 : m.p. 86-88 ºC. Purity of UTK1 measured with PLC was 97.7% Spectral data of B-UTK1ph (3 in Scheme 1): 1 NMR (300 Mz, CDCl 3 : MeD = 10 : 1) : ' (ppm) = 0.86 (3, d, J = 6.0 z), 0.89 (3, s), 0.96 (3, s), 1.03-1.75 (29, m), 1.94 (1, m), 2.08-2.27 (6, m), 2.45 (3, s), 2.74 (1, m), 2.88-2.98 (2, m), 3.11-3.33 (6, m), 4.32-4.38 (2, m), 4.50 (1, m), 4.88 (1, s), 5.01 (1, s), 5.24 (1, br), 5.51 (1, br), 6.06-6.17 (2, br), 6.29 (1, br), 6.34 (1, s), 10.01 (1, s), 12.67 (1, s). ESI-RMS m/z calcd for C 45 70 N 4 Na 8 S [M+Na] + 849.4807, found 849.4769. Spectral data of B-UTK1ox (6 in Scheme 1): 1 NMR (300 Mz, CDCl 3 : MeD = 10 : 1) : ' (ppm) = 0.81 (3, d, J = 7.2 z), 0.83 (3, s), 0.89 (3, s), 0.97-1.89 (32, m), 2.03-2.14(6, m), 2.22 (3, s), 2.64 (1, bd), 2.73 (1, dd, J = 8.4, 12.9 z), 2.84 (1, dd, J = 4.5, 12.9 z), 3.02-3.16 (7, brm), 3.30 (1, m), 4.13 (2, t, J = 6.3 z), 4.22-4.24 (2, m), 4.42 (1, dd, J = 4.5 z, 8.4 z), 4.80 (1, s), 5.02 (1, s), 6.25 (1, s), 6.84-6.91 (2, br), 8.36 (1, s). ESI-RMS m/z calcd for C 48 78 N 6 Na 8 S [M+Na] + 921.5494, found 921.5503. Supplementary References 1. Sawada, M., Kubo, S., Matsumura, K., Takemoto, Y., Kobayashi,., Tashiro, E., Kitahara, T., Watanabe,., and Imoto, M. (2011) Bioorg. Med. Chem. Lett. 21, 1385-1389 2. Ki, S. W., Ishigami, K., Kitahara, T., Kasahara, K., Yoshida, M., and orinouchi, S. (2000) J. Biol. Chem. 275, 39231-39236