Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12‐fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20‐ and 50‐fold in response to insulin and IGF‐1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3‐kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild‐type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase‐2. These experiments demonstrated that activation of PKBalpha by insulin or insulin‐like growth factor‐1 (IGF‐1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF‐1‐stimulated cells by an upstream kinase(s).

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKB␣ was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKB␣ was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32 P labeling and mutagenesis demonstrated that m/p-PKB␣ activity was due to phosphorylation on ). Three mammalian isoforms of PKB have been identified so far, termed PKB␣, -␤, and -␥ (7-9). 2 All three isoforms contain a pleckstrin homology (PH) domain at the N terminus (10), followed by a catalytic domain related to protein kinases A and C, and a C-terminal regulatory region. PKB␣ was found to mediate insulin-and insulin-like growth factor (IGF-1)-induced cellular responses, such as the inhibition of glycogen synthase kinase-3 (11), the stimulation of glucose uptake (12), and the promotion of cell survival by inhibiting apoptosis (Ref. 13; reviewed in Refs. 14 and 15). PKB␣ is the cellular homologue of the oncogene product v-Akt encoded by the AKT8 retrovirus, which induces thymic lymphomas in mice (16). Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKB␣, which adds an N-terminal myristoylation signal to the oncoprotein and could account for its transforming ability (2, 17). Overexpression of PKB␣ or -␤ is associated with some human ovarian, pancreatic, and breast carcinomas (8, 18 -20).PKB␣ is activated by a variety of growth factors and phosphatase inhibitors (5, 6, 21) through a phosphorylation mechanism (21-23). The activation of PKB␣ by insulin or IGF-1 is mediated by phosphorylation of Thr 308 in the catalytic domain and Ser 473 at the C terminus (22). The phosphorylation of both sites is blocked by pretreatment of the cells with the PI3-K inhibitor wortmannin. Substitution of both regulatory sites by aspartic acid residues to mimic phosphorylation by the introduction of a negative charge, produces a constitutively active enzyme (22). This work predicted the existence of an upstream kinase(s) that phosphorylate(s) these sites, and recently a protein kinase activity was identified and purified capable of phosphorylating Thr 308 in the presence of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) (Refs. 24 and 25; reviewed in Ref. 26). The enzyme has therefore been termed 3-phosphoinositide-dependent protein kinase-1 (PDK1).The PH domain of PKB has been reported to play a role in the activation process (6), but PKB activation can also occur in its absence, depending on the agonist and the type of deletion mutants used (21,23,27). The PH domain of PKB binds PtdIns(3,4,5)P 3 and PtdIns(3,4)P...

The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.