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1 Supplementary Materials for The subcellular localization and activity of cortactin is regulated by acetylation and interaction with Keap1 Akihiro Ito, Tadahiro Shimazu, Satoko Maeda, Asad Ali Shah, Tatsuhiko Tsunoda, Shun-ichiro Iemura, Toru Natsume, Takafumi Suzuki, Hozumi Motohashi, Masayuki Yamamoto, Minoru Yoshida* This PDF file includes: *Corresponding author. Published 24 November 2015, Sci. Signal. 8, ra120 (2015) DOI: /scisignal.aad0667 Fig. S1. Acetylation occurs on all seven conserved lysine residues in the repeat domains of human cortactin. Fig. S2. HDAC6 and SIRT2 are the major deacetylases for cortactin in cells. Fig. S3. SIRT1 is localized in the nucleus in A549 and COS-7 cells. Fig. S4. CBP is a major acetylase for cortactin in cells. Fig. S5. CBP is localized in the nucleus in A549 and COS-7 cells. Fig. S6. Specificity of the antibodies for acetylated cortactin. Fig. S7. Specificity of an anti Ac-cttn antibody for immunostaining. Fig. S8. Consensus NES sequences in cortactin and increased acetylation of cortactin upon LMB treatment. Fig. S9. Keap1 binds cortactin independently of actin. Fig. S10. Effect of knockdown of Keap1 or cortactin on cell proliferation. Fig. S11. Keap1 knockdown decreases PMA-induced localization of cortactin to a structure resembling circular dorsal ruffles. Fig. S12. Keap1 knockdown decreases PMA-induced localization of cortactin to structures resembling circular dorsal ruffles. Fig. S13. Acetylation reduces cortical localization of cortactin. Fig. S14. Effects of the Keap1 G430C mutant on repressing the Nrf2 activity and binding to cortactin. Legends for movies S1 to S6 Other Supplementary Material for this manuscript includes the following:

2 (available at Movie S1 (.mov format). Dynamic movement of cortactin in A549 cells stimulated by PMA. Movie S2 (.mov format). Effect of Keap1 knockdown on PMA-induced dynamic movement of cortactin. Movie S3 (.mov format). PMA-induced dynamic movement of cortactin in A549 cells. Movie S4 (.mov format). Effect of TSA and NA on PMA-induced dynamic movement of cortactin. Movie S5 (.mov format). The 7KR mutation rescues PMA-induced dynamic movement of cortactin from inhibition by TSA and NA. Movie S6 (.mov format). Effect of the 7KQ mutation on PMA-induced dynamic movement of cortactin.

3 Supplemental Figure S1. Acetylation occurs on all seven conserved lysine residues in the repeat domains of human cortactin. COS-7 cells were transfected with an expression vector for FLAG-tagged cortactin wild-type (WT), 7KR, or 6KR. In the 6KR constructs, six out of seven conserved lysine residues are replaced with arginine. Cells were then treated for TSA and NA. Proteins immunoprecipitated with anti-flag antibody were analyzed by immunoblotting with anti- AcLys antibody (A) or anti Ac-cttn antibody (B). N = two independent experiments, respectively.

4 Supplemental Figure S2. HDAC6 and SIRT2 are the major deacetylases for cortactin in cells. (A) COS-7 cells were transfected with control or sirna targeting HDAC1, HDAC2, and HDAC3 simultaneously or HDAC6 alone. Cell lysates were immunoblotted with the indicated antibodies. N = two independent experiments. (B) COS-7 cells were transfected with control, SIRT1, SIRT2, or HDAC6 sirna. Proteins immunoprecipitated with anti-cttn antibody were analyzed by immunoblotting. Whole-cell lysates were immunoblotted with anti-sirt1, anti-sirt2, or anti-hdac6 antibody to confirm knockdown efficiency. Anti α-tubulin antibody was used as an internal control. N = four independent experiments.

5 Supplemental Figure S3. SIRT1 is localized in the nucleus in A549 and COS-7 cells. A549 or COS-7 cells were immunostained with anti-cttn antibody along with anti-sirt1 antibody. N = two independent experiments. Scale bar=25 m.

6 Supplemental Figure S4. CBP is a major acetylase for cortactin in cells. (A) Intrinsic enzymatic activity of CBP is required for inducing cortactin acetylation. COS7 cells were transfected with plasmids for expression of HATs, including an enzyme-dead mutant of CBP (CBP mut). Proteins immunoprecipitated from cell lysates using the indicated antibodies were analyzed by immunoblotting. N = two independent experiments. (B) PCAF knockdown does not affect cortactin acetylation. COS-7 cells were transfected with control or sirna oligonucleotides targeting CBP or PCAF mrna, followed by treatment with TSA and NA. The cortactin acetylation was determined by immunoblotting using an anti Ac-cttn antibody. Knockdown efficiency was measured by immunoblotting of whole-cell lysates with an anti-cbp or an anti-pcaf antibody. N = four independent experiments. (C) Reintroduction of CBP restores cortactin acetylation in CBP-knockdown cells. COS7 cells were transfected with control or sirna oligonucleotides targeting 3 -UTR of CBP mrna, followed by tranfection with an empty vector or an expression vector for HA-tagged CBP and treatment with TSA and NA. The cortactin acetylation was determined as described in (B). N = two independent experiments.

7 Supplemental Figure S5. CBP is localized in the nucleus in A549 and COS-7 cells. A549 or COS-7 cells were immunostained with anti-cttn antibody along with anti-cbp antibody. N = two independent experiments. Scale bar=25 m.

8 Cttn-peptide TSA/NA - AcLys 309 Lys AcLys 309 Lys 309 α-ac-cttn α-cttn DAPI Merge 1.2 Relative Ac-cttn level ** ** 1.2 Relative Ac-cttn level AcLys 309 Lys AcLys 309 Lys ** ** Supplemental Figure S6. Specificity of the antibodies for acetylated cortactin. COS-7 cells treated with or without TSA and NA were immunostained with the indicated antibodies. The peptide competition assay was performed as described in Fig. 1D. At least 600 cells per an experiment were analyzed. Data are the means ± S.D. of three independent experiments. **P<0.01. Pictures show representative examples. Scale bar=25 m.

9 Supplemental Figure S7. Specificity of an anti Ac-cttn antibody for immunostaining. COS-7 cells were transfected with control or sirna oligonucleotide targeting Cttn mrna. The cells were then immunostained with the indicated antibodies. N = two independent experiments. Scale bar=25 m.

10 Supplemental Figure S8. Consensus NES sequences in cortactin and increased acetylation of cortactin upon LMB treatment. (A) Two putative NES-like sequences in the SH3 domain of cortactin were named NES1 and NES2. Hydrophobic stretches important for NES function present in NES1 (493- LGITAVAL-500) and NES2 (540-LFPANYVEL-548) are shown with underlines. Sequences of NES mutants (NES1-4A: AGATAAAA and NES2-4A: AAPANYAEA) are shown. (B) Enhancement of cortactin acetylation by LMB. A549 or COS-7 cells treated with LMB for the indicated periods were analyzed by immunoblotting. N = two independent experiments.

11 Supplemental Figure S9. Keap1 binds cortactin independently of actin.

12 (A) Direct interaction between cortactin and Keap1. Purified recombinant cortactin was incubated with GST-fused Keap1, then with glutathione Sepharose beads. Bound proteins were immunoblotted with the indicated antibodies. The nonspecific band was indicated by *. N = two independent experiments. (B) Schematic representation of Keap1 mutants. (C) Identification of the domain responsible for binding to Keap1. FLAG immunoprecipitates from 293T cells expressing the cortactin mutants described in the figure 2B were analyzed by immunoblotting. N = two independent experiments. (D) Identification of the domain in Keap1 responsible for binding to cortactin. FLAG immunoprecipitates from 293T cells expressing the Keap1 mutants described in (B) were analyzed by immunoblotting. N = three independent experiments. (E, F) Keap1 binds cortactin independently of actin. 293T cells were transfected with FLAGtagged cortactin in the presence or absence of latrunculin A. Proteins immunoprecipitated with an anti-flag antibody were analyzed by immunoblotting (E). N = five independent experiments. A549 cells were treated with latrunculin A at the same condition of (E) and the cells were then stained with Phalloidin and DAPI (F). N = two independent experiments. Scale bar=25 m.

13 Supplemental Figure S10. Effect of knockdown of Keap1 or cortactin on cell proliferation. Cells were seeded in a 96-well plate in triplicate after transfection with the indicated sirna oligonucleotides. After 24-h culture, cells were labeled with calcein AM, and the fluorescence was measured (excitation: 485 nm; emission: 530 nm) using a microplate spectrophotometer. Data are the means ± S.D. of three independent experiments.

14 Supplemental Figure S11. Keap1 knockdown decreases PMA-induced localization of cortactin to a structure resembling circular dorsal ruffles. (A) A549 cells transfected with control (sicont) or Keap1 (sikeap1 #2) sirna were serumstarved and then treated with PMA, and cortactin localization was determined by staining with anti-cttn antibody and DAPI. Two representative pictures taken at different magnifications were shown. Cortical localizations of cortactin were indicated by arrows. N = two independent experiments. Scale bar=25 m.

15 Supplemental Figure S12. Keap1 knockdown decreases PMA-induced localization of cortactin to structures resembling circular dorsal ruffles. A549 cells transfected with control (sicont) or Keap1 (sikeap1#1) sirna were cultured under serum-starved conditions. The cells were treated with PMA and then analyzed by immunostaining to detect cortactin and phalloidin staining to label F-actin. N = three independent experiments. Scale bar=25 m.

16 Supplemental Figure S13. Acetylation reduces cortical localization of cortactin. COS-7 cells transfected with FLAG-tagged cortactin wild-type (WT), 7KR mutant, or 7KQ mutant were cultured under serum-starved conditions, and then treated for another with or without TSA and NA. After stimulation with PMA, cells were fixed and stained with an anti- FLAG antibody. Cells expressing FLAG-cortactin were counted and the ratios of the cells with cortical localization were determined. At least 200 cells per an experiment were analyzed. Data are the means ± S.D. of three independent experiments. *P<0.01.

17 Supplemental Figure S14. Effects of the Keap1 G430C mutant on repressing the Nrf2 activity and binding to cortactin. (A) Deficiency of Keap1 G430C mutant in repressing the Nrf2 activity. A549 cells stably infected with an empty retrovirus expression vector (Vec) or a retrovirus encoding HAtagged Keap1 wild type (WT) or G430C mutant (G430C) were immunoblotted with indicated antibodies. N = two independent experiments. (B) Binding of Keap1 G430C mutant to cortactin. 293T cells were transfected with FLAGtagged Keap1 wild-type (WT) or G430C mutant (G430C). Proteins immunoprecipitated with the anti-flag antibody were analyzed by immunoblotting. Whole-cell lysates were also analyzed by immunoblotting to confirm comparable abundance of cortactin. N = two independent experiments.

18 Movies Legends Supplemental Movie S1. Dynamic movement of cortactin in A549 cells stimulated by PMA. GFP-fused cortactin-expressing cells were transfected with control sirna, and live images were recorded after treatment with PMA. One frame was captured per minute. Supplemental Movie S2. Effect of Keap1 knockdown on PMA-induced dynamic movement of cortactin. GFP-cortactin expressing cells were transfected with Keap1 sirna, and live images were recorded after treatment with PMA. One frame was captured per minute. Supplemental Movie S3. PMA-induced dynamic movement of cortactin in A549 cells. Live images were recorded from cells expressing GFP-cortactin wild-type and treated with PMA. One frame was captured per minute. Supplemental Movie S4. Effect of TSA and NA on PMA-induced dynamic movement of cortactin. Cells expressing GFP-cortactin wild-type were pretreated with TSA and NA, and live images were recorded after treatment with PMA. One frame was captured per minute. Supplemental Movie S5. The 7KR mutation rescues PMA-induced dynamic movement of cortactin from inhibition by TSA and NA. Cells expressing GFP-cortactin 7KR mutant were pretreated with TSA and NA, and live images were recorded after treatment with PMA. One frame was captured per minute. Supplemental Movie S6. Effect of the 7KQ mutation on PMA-induced dynamic movement of cortactin. Live images were recorded from cells expressing GFP-cortactin 7KQ mutant and treated with PMA. One frame was captured per minute.