FcγRIIB/SHIP does not inhibit Myr Akt-ER phosphorylation and activation.

Parental A20 cells, or those transfected with Myr Akt-ER or the non-Myr Akt-ER were pretreated with 4-HT and then either left unstimulated, stimulated by cross-linking the BCR (B), or stimulated by co–cross-linking the BCR with the FcgRIIB1 (B+F). Cell lysates were then precipitated with anti-HA antibody and analyzed as described below. (A) In vitro kinase activity toward a GSK3 peptide and the amount of radioactive ³²P incorporation was measured as described in Figure 2. The data are representative of 5 independent experiments using 4 different clones. (B) The cell lysates from the same experiment was also immunoprecipitated with anti-ER antibody and the phosphorylation on Thr³⁰⁸ of Akt construct was assessed by immunoblotting with phosphospecific anti-Akt antibody. (C) The membrane from B was stripped and reprobed with an anti-HA antibody to determine protein levels. The phosphorylated Akt migrated with slower mobility and was seen as a second band only in lanes where phospho-Akt was detectable (seen in B) and correlated with the in vitro Akt kinase activity (seen in A). (D) The left panel represents Myr Akt-ER activity after 10 minutes of simultaneous cell treatment with 4-HT and BCR cross-linking (B) or BCR+FcgRIIB1 (B+F) co–cross-linking. The right panel represents Myr Akt-ER kinase activity after 2 minutes of prestimulation of cells with BCR cross-linking or BCR+FcgRIIB1 co–cross-linking (B+F), followed by 8 minutes of treatment with 4-HT. Baseline radioactivity seen with beads alone without anti-Akt antibody was subtracted from all measurements.