Akt Control Cell Extracts (Rac PKa, PKBa),

Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors and functions in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and also by phosphorylation within the C-terminus at Ser473. Akt functions to promote cell survival by inhibiting apoptosis by means of its ability to phosphorylate and inactivate several targets, including Bad (5), Forkhead transcription factors (6) and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (7). LY294002 is a specific PI3 kinase inhibitor (8). Nonphosphorylated Akt Cell Extracts: Total cell extracts from Jurkat cells, serum starved for 2 hours with 50uM LY294002 treatment during the second hour, serve as a negative control.