RGS18 is phosphorylated on S216 by both PKA and PKGI. (A) Detection of novel phosphoproteins in platelets. Human platelets were treated with 10μM SNP for the indicated time points. Samples were then lysed, separated by SDS-PAGE, and immunoblotted with pS7-Rap1GAP2 Ab. In addition to pS7-Rap1GAP2, phosphorylated S239 of VASP, a prominent band at approximately 30kDa (P30), and a weaker band at 65 kDa (P65) were detected. (B) Phosphorylation of recombinant RGS18 by PKA. Equal amounts of purified GST-RGS18 were incubated with the catalytic subunit of PKA in the presence or absence of ATP. Samples were separated by SDS-PAGE and Western blots were incubated with pS7-Rap1GAP2 Ab. The Ab recognizes the PKA-phosphorylated form of GST-RGS18 (top panel, anti-pS7Rap1GAP2). Protein amounts of GST-RGS18 were verified using monoclonal RGS18 Ab (bottom panel, total RGS18). (C) Protein sequence alignment of the Rap1GAP2 peptide that served as an Ag in the generation of the phosphospecific Ab with a very similar region around S216 on RGS18. (D) Verification of S216 of RGS18 as the phosphorylation site for PKA. wt-RGS18, S216A-RGS18, and S218A-RGS18 GST constructs were incubated with the catalytic subunit of PKA in the presence of [γ-³²P] ATP. Samples were separated by SDS-PAGE and Western blots were exposed to film to detect ³²P signal (top panel). Protein amounts were verified by Western blotting using anti-GST Ab (bottom panel, total RGS18). (E) Verification of specificity of new pS216-RGS18 Ab. GST-RGS18 constructs were incubated with the catalytic subunit of PKA in the presence or absence of ATP. Samples were then subjected to SDS-PAGE and immunoblotted using the pS216-specific Ab (top panel, pS216 RGS18) and monoclonal RGS18 Ab as a loading control (bottom panel, total RGS18). (F) Phosphorylation of endogenous RGS18 in intact human platelets. Platelets were stimulated with PGI₂ (1μM, 5 minutes), forskolin (10μM, 30 minutes), cAMP analog 5,6-di-chlorobenzimidazole riboside-3,5-cyclic monophosphorothioate Sp-isomer (300μM, 30 minutes), sodium nitroprusside (10μM, 10 minutes), GMP analog 8-(4-chlorophenylthio)guanosine-3,5-cyclic monophosphate (300μM, 20 minutes), thrombin (0.1 U/mL, 30 seconds), thromboxane mimetic U46619 (1μM, 1 minute), or ADP (10μM, 1 minute). Samples were then subjected to SDS-PAGE and immunoblotted using the pS216-specific Ab (top panel) and the monoclonal RGS18 Ab as a loading control (bottom panel). Shown are representative data of independent experiments performed at least 3 times.