Fig. 5.
Overexpressed NF1 blunts activation of p87/p110γ, but not p101/p110γ, in murine BMMCs. (A) PKB/Akt phosphorylation (pThr308 and pSer473) in BMMCs. p110γ and HA-p87 or HA-p101 were coexpressed with or without Flag-NF1 in p110γ null BMMCs. Transfected BMMCs were starved for 3 h and then stimulated with 2 μM adenosine (Ade) or 10 ng/mL of murine stem cell factor (SCF) for 2 min. Expression of proteins was verified. NF1-induced attenuation of Ras activity was validated using anti-phosphoMAPK (pMAPK) antibodies. Adenosine-induced p101/p110γ PKB phosphorylation was unchanged, whereas p87/p110γ PKB phosphorylation was depleted in the presence of NF1. At the same time, the phosphorylation of MAPK triggered by adenosine or SCF was affected by NF1. (B) Quantification of adenosine-triggered phosphorylation of PKB (on pSer473 and pThr308) and pMAPK from experiments as shown in (A). Values from NF1 cotransfected cells are expressed as percent of control (p110γ null BMMCs reconstituted with the indicated PI3Kγ complexes). Data are given as mean ± SEM (pSer473, n = 5; pThr308, n = 3; pMAPK, n = 4). *, P < .02.