Figure 2

Dephosphorylation of p85 α S608 does not alter PI kinase activity. A. Dephosphorylation of PI3Kα using different protein phosphatases. Purified, recombinant p110αEE^WT/p85α (200 ng) was assayed for protein (upper panel) or PI (lower panel) kinase activity in the presence of no phosphatase (control), 2 U Antarctic Phosphatase, 10 U Calf Intestinal Alkaline Phosphatase or 40 U Lambda (λ) Protein Phosphatase. Reactions were stopped after 30 minutes using 1 M HCl. B. Lipid kinase activity of dephosphorylated wild-type and mutant PI3Kα. 200 ng purified, recombinant p110αEE^WT/p85α, p110αEE^E545K/p85α or p110αEE^H1047R/p85α was assayed for protein kinase activity (upper panel) or lipid kinase activity using PI as a substrate (lower panel) in the presence or absence of 40 U λ Protein Phosphatase. Reactions were stopped after 40 minutes using 1 M HCl. C. Time courses of phosphorylation of p85α S608 and PI in the presence or absence of a protein phosphatase. 200 ng purified, recombinant p110αEE^WT/p85α (circles), p110αEE^E545K/p85α (triangles) or p110αEE^H1047R/p85α (squares) was assayed for protein kinase activity (upper panel) or lipid kinase activity using PI as a substrate (lower panel) in the presence (closed symbols) or absence (open symbols) of 20 U λ Protein Phosphatase. Reactions were stopped after the indicated times using 1 M HCl.