Figure 4.

Lys-776 and His-917 are required for autophosphorylation on p85α. A, SDS-PAGE analysis and autoradiography for WT and mutant PI3Kα proteins. Autophosphorylation of p85α using radiolabeled ATP as substrate in the absence of PIP₂. The negative control is unphosphorylated p85α in the absence of p110α. The positive control is PTEN-phosphorylated by CK2 kinase. B, autophosphorylation of PI3Kα with and without PIP₂. C, relative quantification of Ser-608 phosphorylation in p85α for WT PI3Kα and mutants by tandem mass spectrometry (MS/MS) of TMT-labeled peptides. D, tandem mass spectrum of TMT labeled peptides showing phosphorylation of Ser-608 (marked by arrows on y15) and reporter ions region (boxed in blue).