Figure 3

Phosphorylation of p85 α S608 does not alter PI kinase activity. 25 μg purified, recombinant p110αEE^WT/p85α, p110αEE^E545K/p85α or p110αEE^H1047R/p85α was incubated with (phosphorylated) or without (mock) 1 mM ATP for 16 hr. Excess ATP was removed by buffer exchange into TBS containing 10 mM 2-mercaptoethanol then phosphorylated or mock phosphorylated PI3Kα was concentrated to approximately 0.5 mg/ml. A. MALDI-MS spectrum from m/z 3400–3750 of a Fe³⁺ IMAC-enriched tryptic digest from phosphorylated (upper spectrum) or mock phosphorylated (lower spectrum) p110αEE^WT/p85α, p110αEE^E545K/p85α and p110αEE^H1047R/p85α. The intensity scale for these spectra were normalised to the peak at m/z 3424.5, which corresponds to a peptide encompassing residues 35–66 from p85α (₃₅GSLVALGFSDGQEAKPEEIGWLNGYNETTGER₆₆) that is not phosphorylated by the protein kinase activity of p110αEE/p85α. Serine 608 from p85α was observed within 2 overlapping peptides of m/z 3483.4 corresponding to residues 594–622 from p85α (₅₉₄LNEWLGNENTEDQYSLVEDDEDLPHHDEK₆₆₂) and m/z 3611.5 corresponding to residues 593–622 from p85α (₅₉₃KLNEWLGNENTEDQYSLVEDDEDLPHHDEK₆₆₂). Incubation with ATP resulted in the appearance of peptides of masses m/z 3563.5 and m/z 3691.6 which are 80 mass units (the mass of a phosphoryl- group) greater than m/z 3483.4 and m/z 3611.5. B. 250 ng of phosphorylated (closed symbols) or mock phosphorylated (open symbols) p110αEE^WT/p85α (circles), p110αEE^E545K/p85α (triangles) or p110αEE^H1047R/p85α (squares) were assayed for PI (lower panel) or protein (upper panel) kinase activities as described above.