Identification of PKC phosphorylation sites of IBtkγ. (A) Schematic representation of the phosphopeptides detected by LC-MS/MS on in vitro phosphorylation of GST-IBtkγ (aa 30-130) (4 μg) with PKCβ (100 ng) followed by tryptic digestion. (B) Picture of IBtkγ N-terminal sequence with the putative PKC phosphorylation sites as predicted by Motifscan software; the PKC phosphorylation sites identified by LC-MS/MS are shown in red circles. (C) GST-IBtkγ (aa 30-130) wild-type or mutants carrying the indicated serine to alanine substitutions (1 μg) were in vitro phosphorylated with PKCmix (25 ng) for 30 minutes in the presence of [γ-³²P]ATP; proteins were analyzed by 8% SDS-PAGE followed by autoradiography and Coomassie blue staining. The densitometry of the phospho-IBtkγ signal was performed in 3 independent experiments, and optical density of phospho-IBtkγ was normalized to Coomassie blue staining. Densitometry of IBtkγ mutants relatively to wild type was expressed as the mean values of 4 independent experiments ± SEM (bottom); the asterisk indicates a statistically significant difference between the IBtkγ mutant and the wild type according to the Student t test: (*P = .0003; **P = .0001; ***P = .0005; ****P = .0001; for n = 4). (D) DeFew cells (2.4 × 10⁷ cells) stably expressing wild-type or mutants FLAG-IBtkγ-IRES-EGFP upon lentiviral transduction were stimulated for 10 minutes with F(ab′)₂ fragments of anti–human IgM (13 μg/mL); cell lysates (1 mg) were immunoprecipitated with anti-phPKCsub Ab and analyzed by 10% SDS-PAGE and Western blotting with anti-FLAG Ab. A representative experiment of 2 independent experiments is shown. (E) Alignment of orthologous IBtkγ amino acid sequences from different species was performed with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/); the PKC sites are indicated in the square and include the S81 and S87 and S90 as identified by LC-MS/MS.