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. 2010 May 4;285(28):21877–21887. doi: 10.1074/jbc.M109.066290

FIGURE 8.

FIGURE 8.

IL-8 induces Rap1 activation through association of CD38 with Epac, PKA, and Rap1. A, IL-8 activates Rap1. LAK cells were treated with 10 pm IL-8 for a specified time, after which a pulldown assay was used to detect the active form of Rap1. B, 8-Br-cADPR blocks IL-8-induced Rap1 activation. The cells were preincubated with 8-Br-cADPR (100 μm) for 20 min before treatment with 10 pm IL-8 for 60 s. C, cADPR but not NAADP activates Rap1. LAK cells were treated with 200 μm cADPR or 50 nm NAADP for 30 s. D, cADPR-induced Rap1 activation was inhibited by a PKA inhibitor. Rap1 activation was determined after the treatment of LAK cells with 100 μm N6-benzoyl-cAMP, 100 μm 8-pCPT-2′-O-Me-cAMP, or 200 μm cADPR for 30 s. The cells were preincubated with Rp-8-Br-cAMPS (100 μm) for 30 min before treatment with 200 μm cADPR. E, Rap1 knockdown in LAK cells exhibits a reduced NAADP formation by IL-8 but not cADPR formation. A total of 60 pmol of siRNA was transfected into 5 × 105 cells using a transfection reagent. After 48 h of transfection, the cells were subjected to immunoblotting with anti-Rap1 pAb (upper panel) and measurement of NAADP/cADPR formation (lower panel) as described under “Experimental Procedures.” The means ± S.E. of three independent experiments are shown. *, p < 0.05, control (Cont) versus IL-8; #, p < 0.01, IL-8 in control siRNA versus IL-8 in Rap1 siRNA. F, IL-8/Epac/PKA-mediated cell migration is blocked in Rap1 knockdown LAK cells. Cells were treated with 10 pm IL-8, 100 μm 8-pCPT-2′-O-Me-cAMP, or 100 μm N6-benzoyl-cAMP. After incubation for 2 h, cell migration was assayed as described under “Experimental Procedures.” The means ± S.E. of three independent experiments are shown. *, p < 0.05, control versus IL-8 or 8-pCPT-2′-O-Me-cAMP or N6-benzoyl-cAMP; **, p < 0.01, IL-8 in control siRNA versus IL-8 in Rap1 siRNA or 8-pCPT-2′-O-Me-cAMP in control siRNA versus 8-pCPT-2′-O-Me-cAMP in Rap1 siRNA or N6-benzoyl-cAMP in control siRNA versus N6-benzoyl-cAMP in Rap1 siRNA. G, IL-8 induces the association of CD38 with Epac, PKA, and Rap1. LAK cells were treated with IL-8 for 30 s. The cells were extracted with a lysis buffer and then subjected to immunoprecipitation (IP) using anti-CD38 mAb. The immunoprecipitated proteins were analyzed by immunoblotting (WB) with anti-CD38 pAb, anti-Epac pAb, anti-PKA pAb, or anti-Rap1 pAb.