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. 2015 Oct 27;290(50):29974–29983. doi: 10.1074/jbc.M115.678003

FIGURE 3.

FIGURE 3.

Phosphorylation of ARHGAP17 by PKA on Ser-702 leads to the dissociation of an ARHGAP17-CIP4 complex. A, ARHGAP17 is a GTPase-activating protein of Rac1. HEK293T cells were transfected with ARHGAP17 (with no tag) and 24 h later endogenous levels of Rac1-GTP were analyzed by pull-down assay followed by SDS-PAGE and Western blotting. Rac1 antibody was used to determine Rac1-GTP (upper panel) and total Rac1 (middle panel) levels and ARHGAP17 antibody was used to determine total ARHGAP17 levels (lower panel). Data are representative of five independent experiments. B, Ser-702 phosphorylation of ARHGAP17 results in the dissociation of CIP4, Toca-1, and PACSIN2 from ARHGAP17 in transfected cells. HEK293T cells expressing EGFP, ARHGAP17-EGFP or ARHGAP17-S702A-EGFP were incubated without or with forskolin (10 μm, 10 min). ARHGAP17 proteins were immunoprecipitated with anti-GFP antibody and co-precipitated proteins were identified by mass spectrometry. Label free quantification (LFQ) intensities of three co-immunoprecipitated proteins CIP4, Toca-1, and PACSIN2 are shown. This experiment was performed once. C, ARHGAP17-S702E mutation mimics phosphorylation-dependent CIP4 dissociation in transfected cells. HEK293T cells expressing EGFP, ARHGAP17-EGFP, ARHGAP17-S702A-EGFP, or ARHGAP17-S702E-EGFP were incubated without or with forskolin (10 μm, 10 min) as indicated. ARHGAP17 proteins were immunoprecipitated with anti-GFP antibody and co-precipitated endogenous CIP4 was analyzed by Western blotting using anti-CIP4 antibody. Anti-ARHGAP17 antibody was used to determine the levels of transfected ARHGAP17. Data are representative of three independent experiments. D, domain composition of ARHGAP17 and CIP4 with the possible interaction site indicated. ARHGAP17 consists of an N-terminal Bin/Amphiphysin/Rvs (N-BAR) domain, a RhoGAP domain, a long proline-rich region with 4 predicted SH3-binding regions and a PDZ-binding motif. Ser-702 is located in the proline-rich region. CIP4 consists of an Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain and a C-terminal Src homology 3 (SH3) domain. E, endogenous ARHGAP17 dissociates from CIP4 in response to PKA activation in platelets. Washed human platelets were incubated without or with PGE1 (0.5 μm, 1 min). Platelets were lysed and purified GST or CIP4-GST was used to pull-down endogenous ARHGAP17. Samples were analyzed by Western blotting using anti-ARHGAP17 antibody to determine precipitated (upper panel) and total ARHGAP17 levels (lower panel). Data are representative of four independent experiments.