PerspectiveSignal Transduction

Update: PtdIns(3,4,5)P3 Gets Its Message Across

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Science  25 Jul 1997:
Vol. 277, Issue 5325, pp. 534
DOI: 10.1126/science.277.5325.534

Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] is clearly a key second messenger in cell regulation, but its mode of action has been somewhat elusive (1). Recent work indicates at least two likely roles—promotion of protein attachment to membranes and activation of protein kinase activity. The current paradigm for understanding PtdIns(3,4,5)P3 action is regulation of the proto-oncogenic serine/threonine protein kinase B (PKB) (also called Akt) (2). On page 567 of this issue of Science and in a recent issue of Current Biology, Stokoe et al. (3) and Alessi et al. (4) provide new insights into PKB activation by phosphorylation (see figure).

Predicting that upstream kinases that activate PKB are regulated by PtdIns(3,4,5)P3, both groups set out to isolate PtdIns(3,4,5)P3-stimulated protein kinases able to phosphorylate PKB at either of the two critical regulatory sites, Thr308 in the T-loop and Ser473 in the carboxyl-terminal regulatory domain. Both groups identified an upstream PKB kinase (called USK, or PKBK as suggested earlier), which phosphorylates Thr308 and partially activates the kinase. The two activities, although similar, may actually represent isoforms of the same kinase family. Alessi et al. (4) termed the activity phospholipid-dependent protein kinase or PDK1. With these purified proteins in hand, the authors then dissected the role of PtdIns(3,4,5)P3 in the activation of USK and subsequently of PKB.

Two roles to play for PtdIns(3,4,5)P3.

Stimulation of receptor tyrosine kinase autophosphorylation promotes recruitment of PI 3-kinase through SRC homology 2 (SH2) domain-phosphotyrosine interactions. Subsequently, PI 3-kinase phosphorylates PtdIns(4,5)P2 to produce the second messenger PtdIns(3,4,5)P3, which localizes PKB and possibly USK to the membrane and alters PKB's conformation. USK can then phosphorylate PKB on Thr308 and partially stimulate its activity. Complete PKB activation requires further phosphorylation of Ser473 by an as yet unidentified kinase. (Inset) Information transfer. Activation of PKB by receptor tyrosine kinase-stimulated PI 3-kinase and PtdIns(3,4,5)P3-stimulated USK.

First, the activity of USK with PKB as substrate is stereospecifically stimulated by PtdIns(3,4,5)P3 [suggesting that it may also contain a pleckstrin homology (PH) domain]. Second, previous work (5) established that the PH domain of PKB binds Ptd(3,4,5)P3 and PtdIns(3,4)P2 with high affinity and that the binding probably causes conformational changes in the kinase. These observations were verified through the use of PKB carrying mutations in its PH domain (Arg25 to Cys and Trp99 to Leu) for the in vitro analysis. Both residues appear critical for full activation of PKB in vivo. Phosphorylation of PKB by USK is abolished by mutations affecting PH domain function (presumably lipid binding) in PKB. As pointed out by Stokoe et al. (3), this suggests that the PH domain of PKB masks the Thr308 residue in the T-loop and that binding of PtdIns(3,4,5)P3 relieves this constraint. Thus, the double role of PtdIns(3,4)P2 in the activation of PKB appears to be resolved by the new findings. Upon addition of this lipid to a coupled kinase assay, PtdIns(3,4,)P2 stimulated the phosphorylation of PKB in a PH domain-dependent manner but did not stimulate USK activity.

The conclusions drawn from these data largely coincide with the earlier predictions (2). PtdIns(3,4,5)P3 appears to be pivotal for the activation process. If in fact USK has a PH domain, there is a strong case for PH domains as signal-dependent membrane adapters (2), probably by mediation of protein kinase complex assembly on the membrane. Within the tethered kinase complex, PtdIns(3,4,5)P3 could also act as an allosteric activator of USK and promote phosphorylation of the downstream target, which itself is “opened up” by lipid binding to its PH domain. A full molecular description of the process will require identification of the predicted Ser473 kinase (at the present rate of discovery, this should be accomplished by the end of 1997).

The new model also indicates how information, that is, extracellular ligand binding to transmembrane receptors, is transferred into the cytoplasm. In this case, PKB activation uses almost the complete spectrum of regulatory mechanisms: tyrosine phosphorylation, protein recruitment, phosphorylation of lipids to act as second messengers, allosteric activation, and serine/threonine phosphorylation (see inset). Regulation of the regulators will obviously prove to be complex—but important in life and death decisions, as emphasized by the recent identification of an oncogenic form of the phosphoinositide 3-kinase (PI 3-kinase) (6).

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