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First published online 24 July 2008
doi: 10.1242/jcs.028647

Journal of Cell Science 121, 2635-2642 (2008)
Published by The Company of Biologists 2008
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Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF

Reina E. Itoh1, Etsuko Kiyokawa1, Kazuhiro Aoki1, Teruko Nishioka1, Tetsu Akiyama2 and Michiyuki Matsuda1,*

1 Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan
2 Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan


Figure 1
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  		Fig. 1. Effect of Sos, Vav2, and Tiam1 depletion on EGF-induced Rac1 activation. (A) Schematic, showing the current view of regulation of Rac1 by EGF. (B) A431 cells were transfected with either Sos1- and Sos2-specific siRNAs, Vav2-specific siRNA or Tiam1-specific siRNA. Forty-eight hours after transfection, cells were lysed and subjected to immunoblot analysis using antibodies against Sos1, Sos2, Vav2 and Tiam1. (C) A431 cells were transfected with siRNA as indicated. One day after siRNA transfection, cells were further transfected with pRaichu-Rac1. After serum starvation for 6 hours, cells were stimulated with 25 ng/ml EGF and examined for Rac1 activity by FRET microscopy as described in the text. Averages of the fold increase after EGF addition over the control cells are given + s.d. Numbers of cell examined under each condition were as following: control, n=85; Sos1,2 KD, n=13; Vav2 KD, n=9; Tiam1 KD, n=90. *P<0.05, significant difference as compared with the control (Student's t-test). (D) Control and knockdown cells were starved for 6-12 hours, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and examined for active Rac1 by pull-down assay. Experiments were repeated at least three times, and average values of fold increase over the control cells are given + s.d. *P<0.05, significant difference as compared with the control (Student's t-test).

 

Figure 2
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  		Fig. 2. Effect of Asef deficiency on EGF-induced activation of Rac1. (A) A431 cells were transfected with pSuper-Luc or pSuper-Asef. After selection with puromycin, the expression level of Asef mRNA was examined by RT-PCR (left). (B) To confirm the the efficiency of knockdown, cells transfected with a control shRNA expression vector and an Asef expression vector were analyzed by immunoblotting (right). Signal intensities of each band were quantified and are shown below each band as a relative strength to the control (in %). (C) A431 cells were transfected with control siRNA or Asef siRNA. Two days later, knockdown efficiency was examined by immunoblotting. Signal intensities of each band were quantified and are shown below each band as a relative strength to the control (in %) in B and C. (D-F) A431 cells were transfected with an empty pSuper vector (control) or pSuper-Asef. After puromycin selection, cells were further transfected with pRaichu-Rac1. After 3-6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF. (D) Representative FRET images at the indicated time points are shown in the intensity-modulated display mode. (E) Normalized FRET:CFP ratios as described in the text. (F) From the peak values of the normalized ratio, the effect of Asef knockdown on EGF-induced Rac1 activation was calculated as described in the legend to Fig. 1D. The error bars indicate + s.d. Numbers of cells under each condition were as follows: control, n=13; Asef KD, n=14. *P<0.05, significant difference as compared with control (Student's t-test). (G) Control and Asef knockdown cells were starved for 6-12 hours, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and were examined for active Rac1 by pull-down assay. Experiments were repeated at least three times, and average values of the fold increase over control cells are given + s.d. *P<0.05, significant difference as compared with the contro (Student's t-test).

 

Figure 3
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  		Fig. 3. Effect of depletion of GEFs on EGF-induced Cdc42 activation. (A) Cells were prepared as described for Figs 1 and 2, except that pRaichu-Cdc42 was transfected instead of pRaichu-Rac1. After serum starvation, cells were stimulated with 25 ng/ml EGF and examined for Cdc42 activity by FRET microscopy as described in the text. The averages of the fold increase over the control cells are shown + s.d. Numbers of cells examined for each condition were as follows: control siRNA, n=23; Vav2 KD, n=6; Tiam1 KD, n=13; control shRNA, n=6; Asef KD, n=3. *P<0.05, significant difference as compared with the control by t-test analysis (Student's t-test). (B) Control and knockdown cells were starved for 6-12 h, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and were examined for active Cdc42 by pull-down assay. Experiments were repeated at least three times, and average values of the fold increase over the mock-treated cells are shown + s.d. *P<0.05, significant difference as compared with the control by t-test analysis (Student's t-test).

 

Figure 4
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  		Fig. 4. EGF-induced tyrosine phosphorylation of Asef. A431 cells expressing HA-Asef, HA-Tiam1-C1199 or HA-Asef2 were serum starved for 24 hours and stimulated with 100 ng/ml EGF for 2 minutes or left unstimulated. Immunoprecipitates and total cell lysates were immunoblotted using anti-phosphotyrosine (pTyr) or anti-HA antibody.

 

Figure 5
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  		Fig. 5. Identification of the principal phosphorylation site of Asef. (A) Domain structures of Asef and Asef2. ABR, APC-binding region; SH3, Src-homology 3; DH, Dbl-homology; PH, pleckstrin homology. (B) Alignment of amino-terminal sequences of Asef and Asef2. Tyr94 and Tyr104 are shown in bold, and the ABR and SH3 domains are underlined and boxed, respectively. The NcoI recognition site used to construct Asef2-Asef1 and Asef1-Asef2 mutants (Asef2/1 and Asef/2, respectively) is shown. (C,D) HA-tagged proteins were expressed transiently in A431 cells and immunoprecipitated with anti-HA antibody before or after 25 ng/mL EGF stimulation for 2 minutes. Immunoprecipitates and total-cell lysates were resolved by SDS-PAGE and probed with anti-phosphotyrosine (pTyr) or anti-HA antibody. (E) A431 cells expressing HA-tagged Asef were stimulated with or without 100 ng/mL EGF for 2 minutes. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the antibodies indicated above each lane. In the case of anti-phosphorylated-Y94Asef ({alpha}-pY94Asef), phenylphosphate was included in the reaction buffer to reduce the signals of non-specific anti-phosphotyrosine antibody. (F) HA-tagged constructs were expressed in A431 cells and immunoprecipitated with anti-HA antibody as in C. Immunoprecipitates and total cell lysates were analyzed using the antibodies as indicated.

 

Figure 6
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  		Fig. 6. Role of Src-family tyrosine kinases on the Asef phosphorylation. (A) HA-Asef-expressing A431 cells were stimulated with 25 ng/ml EGF for 2 minutes. When indicated, cells were pre-treated with 4 µM PP2 for 30 minutes or with 10 µM STI-571 for 2 hours. HA-Asef was immunoprecipitated and analyzed using anti-phosphotyrosine (pTyr) or anti-HA antibodies. (B) HA-Asef expressed alone or co-expressed with v-Src, c-Src-Y537F mutant or c-Abl, was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting using anti-phosphotyrosine (pTyr) or anti-HA antibodies. (C) A431 cells were transfected with HA-Asef (WT) or HA-AsefY94F (YF) in the presence or absence of the c-Src Y527F mutant. Asef was immunoprecipitated and analyzed using antibody against phospho-Tyr94 Asef (anti-pY94Asef) or anti-HA antibody. (D) HA-Asef and HA-AsefY94F expressed in 293F cells were immunoprecipitated using anti-HA antibody, phosphorylated using purified Src protein, and analyzed by immunoblotting as indicated. (E) HA-Asef-expressing A431 cells were stimulated with 50 ng/ml EGF for the indicated period and analyzed as in C.

 

Figure 7
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  		Fig. 7. Detection of tyrosine-phosphorylated Asef at lamellipodia and membrane ruffles. (A,B) A431 cells expressing HA-Asef (A) or HA-AsefY94F (B) were serum starved for 24 hours and then stimulated with 25 ng/ml EGF for 2 minutes. Cells fixed and stained using antibody against phospho-Tyr94 Asef (anti-pY94Asef) or anti-HA antibody were analyzed using a confocal microscope. (C,D) Fluorescence intensities of HA-Asef-WT (C) or HA-Asef-Y94F (D) are plotted along the red lines in A and B.

 

Figure 8
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  		Fig. 8. Asef phosphorylation at Tyr94 has an essential role in EGF-induced activation of Rac1 and Cdc42. (A) A431 cells were transfected with vectors expressing wild-type Asef (WT) and siRNA-resistant Asef (WTR) were harvested at 48 hours after transfection and analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. pSuper-Asef is an shRNA expression vector targeting Asef; pERedNLS-3HA-Asef-WT is a wild-type Asef expression vector; and pERedNLS-3HA-Asef-WTR is a wild-type Asef expression vector carrying RNAi-resistant nuleotide substitutions. R, siRNA-resistant expression plasmids. (B) A431 cells were transfected with expression vectors as indicated below each bar (WTR, pERedNLS-3HA-Asef-WTR; Y94FR, pERedNLS-3HA-Asef-Y94FR). After selection with puromycin, cells were transfected with pRaichu-Rac1 for 24 hours. After 3-6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF and analyzed as described for Figs 2 and 3. Numbers of cells examined for each condition were as follows: control, n=22; Asef KD, n=21; Asef KD and Asef WTR; n=11; Asef KD and Asef Y94FR, n=11. *P<0.05, significant difference over the control cells (Student's t-test). *P<0.05. (C) Experiments were performed as in B except that pRaichu-Cdc42 was used instead of pRaichu-Rac1. Numbers of cells examined for each condition were as follows: control, n=10; Asef KD, n=10; Asef KD and Asef WTR, n=14; Asef KD and Asef Y94FR, n=11.

 

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© The Company of Biologists Ltd 2008