Figure 2
Figure 2. RGS18 interacts with 14-3-3 via S218 in a phosphorylation-dependent manner. (A) Alignment of RGS18 protein sequences from different species using ClustalW2 multiple sequence alignment. A conserved region comprising 7 amino acids is marked with a black border. This region contains both the identified PKA and PKGI phosphorylation site S216 and S218 and a 14-3-3–binding site. Uniprot accession numbers of sequences are: mouse, Q9NS28; rat, Q4L0E8; human, Q9NS28; frog, Q66IM3; and zebrafish, Q08BE2. (B) Endogenous interaction of 14-3-3 with RGS18 in platelets. Washed human platelets were treated without (−) or with either forskolin (10μM, 30 minutes) or thrombin (0.1 U/mL, 30 seconds). Platelets were then lysed and subjected to immunoprecipitation with rabbit anti-RGS18 Ab. As control, platelets were stimulated with thrombin, lysed, and subjected to immunoprecipitation with preimmune serum (ctr thrombin). After SDS-PAGE, Western blots were incubated with mouse anti–14-3-3γ Ab to detect total (middle panel, total 14-3-3γ) and precipitated 14-3-3γ (top panel, 14-3-3γ). Total RGS18 amounts were verified using rabbit anti-RGS18 Ab (bottom panel, total RGS18). Densitometric analysis of the blots of 3 independent experiments confirmed that the difference between control and RGS18 IP in the presence of thrombin was statistically significant (P < .05, data not shown). (C) Interaction of 14-3-3 and RGS18 in transfected cells. FLAG-RGS18 constructs were expressed in HEK293T cells. wt-RGS18, S216A-RGS18, S216E-RGS18, and S218A-RGS18 lysates were subjected to pull-down assays with purified GST-14-3-3γ. As a control, a separate FLAG-wt-RGS18 lysate was subjected to a pull-down assay using GST alone. After SDS-PAGE, Western blots of pull-downs and lysates were incubated with FLAG-Ab to detect precipitated RGS18 (top panel, RGS18) and total RGS18 (bottom panel, total RGS18). (D) Phosphorylation-dependent interaction of RGS18 and 14-3-3 in platelets. Platelets were lysed and subjected to pull-down assays with purified GST-14-3-3γ. Pull-downs were then incubated with λ protein phosphatase for 60 minutes at 30°C according to the manufacturer's protocol. After SDS-PAGE, Western blots of totals and pull-downs were incubated with mouse anti-RGS18 Ab to detect precipitated (top panel, RGS18) and total RGS18 (bottom panel, total RGS18). (E) Phospho-S218–dependent interaction of RGS18 and 14-3-3 in platelets. Platelets were lysed and supplemented with either none or 100μM dephospho (TNLRRRSRSFTVN) or phospho-peptide (TNLRRRSR(pS)FTVN), mimicking the 14-3-3 interaction site. Lysates were subsequently subjected to pull-down assays with purified GST-14-3-3γ. After SDS-PAGE, Western blots of lysates and pull-downs were incubated with mouse anti-RGS18 to detect precipitated (top panel, RGS18) or total (bottom panel, total RGS18) RGS18. Panels B through E are representative data of independent experiments performed at least 3 times.

RGS18 interacts with 14-3-3 via S218 in a phosphorylation-dependent manner. (A) Alignment of RGS18 protein sequences from different species using ClustalW2 multiple sequence alignment. A conserved region comprising 7 amino acids is marked with a black border. This region contains both the identified PKA and PKGI phosphorylation site S216 and S218 and a 14-3-3–binding site. Uniprot accession numbers of sequences are: mouse, Q9NS28; rat, Q4L0E8; human, Q9NS28; frog, Q66IM3; and zebrafish, Q08BE2. (B) Endogenous interaction of 14-3-3 with RGS18 in platelets. Washed human platelets were treated without (−) or with either forskolin (10μM, 30 minutes) or thrombin (0.1 U/mL, 30 seconds). Platelets were then lysed and subjected to immunoprecipitation with rabbit anti-RGS18 Ab. As control, platelets were stimulated with thrombin, lysed, and subjected to immunoprecipitation with preimmune serum (ctr thrombin). After SDS-PAGE, Western blots were incubated with mouse anti–14-3-3γ Ab to detect total (middle panel, total 14-3-3γ) and precipitated 14-3-3γ (top panel, 14-3-3γ). Total RGS18 amounts were verified using rabbit anti-RGS18 Ab (bottom panel, total RGS18). Densitometric analysis of the blots of 3 independent experiments confirmed that the difference between control and RGS18 IP in the presence of thrombin was statistically significant (P < .05, data not shown). (C) Interaction of 14-3-3 and RGS18 in transfected cells. FLAG-RGS18 constructs were expressed in HEK293T cells. wt-RGS18, S216A-RGS18, S216E-RGS18, and S218A-RGS18 lysates were subjected to pull-down assays with purified GST-14-3-3γ. As a control, a separate FLAG-wt-RGS18 lysate was subjected to a pull-down assay using GST alone. After SDS-PAGE, Western blots of pull-downs and lysates were incubated with FLAG-Ab to detect precipitated RGS18 (top panel, RGS18) and total RGS18 (bottom panel, total RGS18). (D) Phosphorylation-dependent interaction of RGS18 and 14-3-3 in platelets. Platelets were lysed and subjected to pull-down assays with purified GST-14-3-3γ. Pull-downs were then incubated with λ protein phosphatase for 60 minutes at 30°C according to the manufacturer's protocol. After SDS-PAGE, Western blots of totals and pull-downs were incubated with mouse anti-RGS18 Ab to detect precipitated (top panel, RGS18) and total RGS18 (bottom panel, total RGS18). (E) Phospho-S218–dependent interaction of RGS18 and 14-3-3 in platelets. Platelets were lysed and supplemented with either none or 100μM dephospho (TNLRRRSRSFTVN) or phospho-peptide (TNLRRRSR(pS)FTVN), mimicking the 14-3-3 interaction site. Lysates were subsequently subjected to pull-down assays with purified GST-14-3-3γ. After SDS-PAGE, Western blots of lysates and pull-downs were incubated with mouse anti-RGS18 to detect precipitated (top panel, RGS18) or total (bottom panel, total RGS18) RGS18. Panels B through E are representative data of independent experiments performed at least 3 times.

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