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First published online June 18, 2008
doi: 10.1242/10.1242/jcs.028415

Journal of Cell Science 121, 2177-2185 (2008)
Published by The Company of Biologists 2008
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Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion

Ursula Rescher1,*,{ddagger}, Carsten Ludwig1,*, Vera Konietzko1,*, Alexei Kharitonenkov2 and Volker Gerke1,{ddagger}

1 Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, University of Muenster, 48149 Muenster, Germany
2 Biotechnology Discovery Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA


Figure 1
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  		Fig. 1. Insulin induces rapid and sustained tyrosine phosphorylation of annexin A2 in BHK cells stably expressing the human insulin receptor. (A) Time course of insulin-induced tyrosine phosphorylation of BHK-IR cell proteins. Serum-starved BHK-IR cells were treated with 100 nM insulin for the indicated times and total cellular lysates were probed by immunoblotting with anti-phosphotyrosine antibodies. Arrows indicate the positions of the insulin receptor β-subunit (IRβ) and the heavily phosphorylated 36 kDa protein (pp36). (B) The tyrosine-phosphorylated polypeptide of 36 kDa is annexin A2. Lysates of cells stimulated for the indicated periods of time with 100 nM insulin were subjected to immunoprecipitation (IP) with the monoclonal anti-annexin A2 antibody HH7. The precipitated protein was immunoblotted (WB) for phosphotyrosine ({alpha}-pY) and the blot subsequently reprobed with anti-annexin A2 antibodies ({alpha}-Anxa2). c, control lane (lysates were incubated with beads alone). (C) Annexin A2 phosphorylation increases during the first 3 hours of insulin treatment. Serum-starved cells were stimulated for the indicated times with 100 nM insulin. The intensity of the pp36 band was measured by densitometric scanning and is given as arbitrary units (mean values ± s.e.m.).

 

Figure 2
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  		Fig. 2. Insulin induces massive actin rearrangements in BHK-IR cells. (A) Insulin-induced detachment of BHK-IR cells. Cells grown in 24-well plates were serum-starved overnight and stimulated with 100 nM insulin for 4 hours. Detached cells in the supernatant were counted. Bar graphs represent mean ± s.e.m. from a representative experiment with five parallel samples. *P<0.05. (B) Insulin-induced changes in the actin cytoskeleton of BHK-IR cells. Serum-starved cells grown on coverslips were stimulated for 30 or 60 minutes with 100 nM insulin, fixed and stained for F-actin with TRITC-phalloidin. (C) y-z and x-y sections of representative noninduced cells or cells induced for 30 minutes with insulin are shown, with their typical F-actin stain. Note the increase in height and the concurrent appearance of actin domes or sheaths in the insulin-treated cells. (D) Serum-starved cells were stimulated for 60 minutes with 100 nM insulin, fixed, stained for F-actin with TRITC-phalloidin and co-stained with anti-annexin-A2 (Anxa2) or anti-phosphotyrosine (pY) antibodies, respectively. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Annexin A2 depletion inhibits insulin-induced actin rearrangements. (A) Downregulation of annexin A2 does not affect the general tyrosine phosphorylation in BHK-IR cells. Cells were nontransfected, transfected with control (mock) or annexin A2-specific siRNAs, serum-starved and insulin-stimulated for 30 minutes as indicated. Total cellular lysates were then immunoblotted for phosphotyrosine ({alpha}-pY). The blot was stripped and reprobed for the IRβ-subunit ({alpha}-IRβ) and annexin A2 ({alpha}-Anxa2). (B) Effect of RNAi-mediated downregulation of annexin A2 on insulin-induced actin rearrangements. BHK-IR cells, grown on coverslips, were mock transfected or transfected with annexin A2 siRNA, serum-starved and either nonstimulated or stimulated with 100 nM insulin for 30 minutes. The fixed cells were stained for F-actin and morphological changes in the actin cytoskeleton (appearance of the `insulin-responsive' actin phenotype) were quantified as described in the Materials and Methods. (C) Re-expression of annexin A2 in annexin-A2-depleted cells restores the ability to insulin-dependently remodel F-actin. Western blotting of total cell lysates shows comparable levels of annexin A2-GFP expression in annexin A2 siRNA and mock-treated cells. GFP-expressing cells remained unable to undergo insulin-induced F-actin changes, whereas cells re-expressing annexin A2-GFP responded similar to nondepleted cells and displayed the insulin-induced actin phenotype. **P<0.0005.

 

Figure 4
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  		Fig. 4. The Rho/ROCK pathway is involved in mediating insulin-induced morphological changes in BHK-IR cells. (A) The active RhoA mutant RhoQ63L induces an insulin-like phenotype. BHK-IR cells were transfected with a Myc-tagged RhoQ63L construct and co-stained with anti-Myc-antibodies and TRITC-phalloidin or anti-annexin-A2 antibodies. Note the appearance of the typical `insulin phenotype', i.e. cell rounding and the formation of actin domes upon RhoQ63L expression. Scale bars: 10 µm. (B) Rho/ROCK activity is required for insulin-induced morphological changes. Serum-starved cells grown on coverslips were left untreated or preincubated either with the cell-permeable Rho-inhibiting C3 exotoxin or with two different, structurally unrelated ROCK inhibitors, Y27632 and H-1152, prior to insulin stimulation for 30 minutes. The fixed cells were stained for F-actin and morphological changes in the actin cytoskeleton were quantified. **P<0.0005.

 

Figure 5
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  		Fig. 5. Insulin-triggered Rho activation requires annexin A2. (A) BHK-IR cells were transfected with either control (mock) or annexin A2 siRNA or with the RhoQ63L expression construct. Cells were treated with 100 nM insulin as indicated, fixed and probed for Rho GTP loading state with GST-RBD as described in the Materials and Methods. Bound GST-RBD was detected by staining with anti-GST-antibody. F-actin was visualized with TRITC-phalloidin. Note the insulin-induced increase in GST-RBD binding that is not seen in annexin-A2-downregulated cells. Scale bars: 10 µm. (B) Bound GST-RBD was quantified using the MetaMorph software (mean values ± s.e.m.). *P<0.05.

 

Figure 6
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  		Fig. 6. The phospho-mimicking annexin A2 mutant Anxa2-Y23E-GFP induces morphological changes. BHK-IR cells grown on coverslips were transfected with either wild type (Anxa2-GFP) or mutant Y23E annexin A2 (Anxa2-Y23E-GFP) and stained for F-actin with TRITC-phalloidin. x-y, x-z and y-z sections of representative transfected and nontransfected cells are shown with their respective typical F-actin stain. Note the increase in height and the concomitant appearance of actin domes or sheaths in cells expressing Anxa2-Y23E-GFP. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. ROCK inhibition abrogates the dominant effect of the phospho-mimicking annexin A2 mutant. (A) BHK-IR cells were transfected with the phospho-mimicking mutant Anxa2-Y23E-GFP and either left untreated or were treated for 1 hour with 20 µM of the ROCK inhibitor Y27632. Cells were then fixed and stained for F-actin. Note the induction of the `insulin-responsive' actin phenotype in Anxa2-Y23E-GFP expressing cells (lower panels), which is abrogated by ROCK inhibition (upper panels). Scale bars, 10 µm. (B) Several hundred cells expressing Anxa2-Y23E-GFP were inspected and the relative number of cells showing the normal F-actin distribution (rescued phenotype) was quantified for experiments carried out in the presence (+) or absence (–) of Y27632. **P<0.0005.

 

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