Mechanism of Activation of PKB

The three isoforms of PKB (PKBa, PKB0, and PKBy) possess high sequence identity and are widely expressed in human tissues [17]. Stimulation of cells with agonists that activate PI 3-kinase induce a large activation of PKB isoforms within a few minutes. The activation of PKB is downstream of PI 3-kinase, as inhibitors of PI 3-kinase such as wortmannin or LY294002, or the over-expression of a dominant-negative regulatory subunit of PI 3-kinase inhibit the activation of PKB in cells by virtually all agonists tested [24-26]. Over-expression of a constitutively active mutant of PI 3-kinase induces PKB activation in unstimulated cells [27],

Figure 1 Overview of the PI 3-kinase signaling pathway. Insulin and growth factors induce the activation of PI 3-kinase and generation of PtdIns(3,4,5)P3. In addition to leading to the activation of PKB/Akt, S6K, SGK, and atypical PKC isoforms such as PKCZ, PtdIns(3,4,5)P3 also recruits a number of other proteins (outlined in the text) to the plasma membrane to trigger the activation of non-PDKl/AGC-kinase-dependent signaling pathways. Key challenges for future experiments are not only to define the specific cellular roles of the individual AGC kinase but also to understand the function and importance of other branches of signaling pathways activated by PI 3-kinase.

Figure 1 Overview of the PI 3-kinase signaling pathway. Insulin and growth factors induce the activation of PI 3-kinase and generation of PtdIns(3,4,5)P3. In addition to leading to the activation of PKB/Akt, S6K, SGK, and atypical PKC isoforms such as PKCZ, PtdIns(3,4,5)P3 also recruits a number of other proteins (outlined in the text) to the plasma membrane to trigger the activation of non-PDKl/AGC-kinase-dependent signaling pathways. Key challenges for future experiments are not only to define the specific cellular roles of the individual AGC kinase but also to understand the function and importance of other branches of signaling pathways activated by PI 3-kinase.

as does deletion of the PTEN phosphatase which also results in increased cellular levels of PtdIns(3,4,5)P3 [28-32].

All PKB isoforms possess an N-terminal pleckstrin homology (PH domain) that interacts with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 followed by a kinase catalytic domain and then a C-terminal tail. Stimulation of cells with agonists that activate PI 3-kinase induces the translocation of PKB to the plasma membrane, where PtdIns(3,4,5)P3 as well as PtdIns(3,4)P2 are located and, consistent with this, translocation of PKB is prevented by inhibitors of PI 3-kinase or by the deletion of the PH domain of PKB [33-35]. These findings strongly indicate that PKB interacts with PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 in vivo. The binding of PKB to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 does not activate the enzyme but instead recruits PKB to the plasma membrane where it becomes phosphorylated at two residues at this location, namely Thr308 and Ser473. Inhibitors of PI 3-kinase and dominant-negative PI 3-kinase prevent phosphorylation of PKB at both residues following stimulation of cells with insulin and growth factors [17]. Thr308 is located in the T-loop (also known as activation loop) between subdomains VII and VIII of the kinase catalytic domain, situated at the same position as the activating phosphorylation sites found in many other protein kinases. As discussed later, Ser473 is located outside of the catalytic domain in a motif that is present in most AGC kinases and which has been termed the hydrophobic motif. The phosphorylation of PKB a at both Thr308 or Ser473 is likely to be required to activate PKBa maximally, as mutation of Thr308 to Ala abolishes PKBa activation, whereas mutation of Ser473 to Ala reduces the activation of PKBa by approximately 85%. The mutation of both Thr308 and Ser473 to Asp (to mimic the effect of phos-phorylation by introducing a negative charge) increases PKBa activity substantially in unstimulated cells, and this mutant cannot be further activated by insulin [3]. Attachment of a membrane-targeting domain to PKBa results in it becoming highly active in unstimulated cells and induces a maximal phosphorylation of Thr308 and Ser473 [33,36]. These observations indicate that recruitment of PKB to the membrane of unstimulated cells is sufficient to induce the phosphorylation of PKBa at Thr308 and Ser473. Furthermore, there must be sufficient basal levels of PtdIns(3,4,5)P3/PtdIns(3,4)P2, T308 kinase, and Ser473 kinase located at the membrane to stimulate phosphoryla-tion and activation of membrane-targeted PKB. PKBP and PKBy are activated by phosphorylation of the equivalent residues in their T-loops and hydrophobic motifs [37,38].

PKB Is Activated by PDK1

A protein kinase was purified [39,40] and subsequently cloned [41,42] that phosphorylated PKBa at Thr308 only in the presence of lipid vesicles containing PtdIns(3,4,5)P3 or PtdIns(3,4)P2. Because of these properties it was named 3-phosphoinositide-dependent protein kinase 1 (PDK1) and is composed of an N-terminal catalytic domain and a C-terminal PH domain which, like that of PKB, interacts with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 [42,43]. The activation of PKB by PDK1 is stereospecific for the physiological D-enantiomers of these lipids, and neither PtdIns(4,5)P2 nor any inositol phospholipid other than PtdIns(3,4)P2 can replace PtdIns(3,4,5)P3 in the PDK1-catalyzed activation of PKB [39,42].

Although co-localization of PKB and PDK1 at the plasma membrane through their mutual interaction with 3-phosphoinositides is likely to be important for PDK1 to phosphorylate PKB, the binding of PKB to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 is also postulated to induce a conformational change in PKB, exposing Thr308 for phosphorylation by PDK1. This conclusion is supported by the observation that in the absence of 3-phosphoinositides, PDK1 is unable to phosphorylate wild-type PKB under conditions where it is able to efficiently phosphorylate a mutant form of PKB that lacks its PH domain, termed APH-PKB [40,41]. Consistent with this, a PKB mutant in which a conserved Arg residue in the PH domain is mutated to abolish the ability of PKB to bind PtdIns(3,4,5)P3 cannot be phosphorylated by PDK1 in the presence of lipid vesicles containing PtdIns(3,4,5)P3 [40]. Moreover, artificially promoting the interaction of PDK1 with wild-type PKB and APH-PKB by the attachment of a high-affinity PDK1 interaction motif to these enzymes is sufficient to induce maximal phosphoryla-tion of Thr308 in APH-PKB but not in wild-type PKB in unstimulated cells [44].

More recently, the three-dimensional structure of the isolated PH domain of PKB complexed with the head group of

PtdIns(3,4,5)P3 has been solved [45]. Interestingly, the structure of the PH domain of PKB complexed to the inositol head group of PtdIns(3,4,5)P3 revealed that the 3- and the 4-phosphate groups form numerous interactions with specific basic amino acids in the PKB PH domain, but in contrast the 5-phosphate group does not make any significant interaction with the protein backbone and is solvent exposed, thus providing the first structural explanation of why PKB interacts with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity [45].

The interaction of PDK1 with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 is thought to be the primary determinant in enabling PDK1 and PKB to colocalize at membranes and permitting PDK1 to phosphorylate PKB efficiently. These conclusions are supported by the finding that the rate of activation of PKBa by PDK1 in vitro, in the presence of lipid vesicles containing PtdIns(3,4,5)P3, is lowered considerably if the PH domain of PDK1 is deleted. Furthermore, the mutant of PKB that lacks its PH domain is also a very poor substrate for PDK1, compared to wild-type PKB, as it is unable to interact with lipid vesicles containing PtdIns(3,4,5)P3.

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