Research CommentariesSignal Transduction

Lipid-Regulated Kinases: Some Common Themes at Last

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Science  30 Jan 1998:
Vol. 279, Issue 5351, pp. 673-674
DOI: 10.1126/science.279.5351.673

Kinases are enzymes that add phosphates to small molecules or other enzymes, creating active signaling molecules or turning enzymes on or off. Cells use this device over and over again to change their internal biochemistry in response to signals from the outside. One class of these kinases [phosphoinositide 3-kinases (PI 3-kinases)] phosphorylate lipids to form the second messenger phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] in response to a very wide range of extracellular stimuli. PtdIns(3,4,5)P3 acts on pathways that control cell proliferation, cell survival, and metabolic changes—often through two different protein kinases, p70 ribosomal protein S6 kinases (p70s6k) and protein kinase B (PKB). Until now the way PtdIns(3,4,5)P3 regulates these two kinases has appeared to be very different, with each new advance adding to the impression that they had very little in common. However, two reports in this issue of Science on pages 707 and 711 (1, 2) and two recent reports in Current Biology (3, 4) add considerably to the picture of how these kinases are regulated, and in so doing reveal surprising similarities in their control mechanisms. The same regulatory themes likely apply to many protein kinases, significantly simplifying the task of understanding their regulation.

The ability of PKB, also known as Akt, to phosphorylate its substrates is increased after the cell is exposed to growth factors and insulin, and this increase occurs via a pathway that includes PI 3-kinase. In addition to its kinase domain, PKB contains an amino-terminal pleckstrin homology (PH) domain, to which the PtdIns(3,4,5)P3 signaling molecule binds (5). In addition, PKB itself is phosphorylated when PtdIns(3,4,5)P3 is present by two different protein kinases, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which phosphorylates Thr308 in the activation loop of the kinase domain of PKB, and PDK2, which phosphorylates Ser473 near the carboxyl-terminal (6, 7). Phosphorylation of these two sites is sufficient to fully activate PKB.

Similarities revealed for two key kinases, PKB and p70s6k.

Upon binding the messenger PtdIns(3,4,5)P3, PKB is translocated to the plasma membrane, and PH domain masking of Thr308 is relieved. PKB is then sequentially phosphorylated at Thr308 by PDK1, which is constitutively active, and at Ser473 by PDK2, which is PtdIns(3,4,5)P3 dependent, to yield a fully activated kinase. p70s6k is phosphorylated initially by proline-directed kinases including MAP kinase in the autoinhibitory domain, then by a PtdIns(3,4,5)P3-dependent kinase at Thr389 to generate a conformation where Thr229 is accessible for phosphorylation by PDK1 to yield a fully active enzyme.

Stephens et al. (2) and Alessi et al. (3) now report the cloning of PDK1 and clarification of its function. PDK1 has a kinase domain that is distantly related to that of PKB and, like PKB, also contains a PH domain that binds to PtdIns(3,4,5)P3 very tightly. However, unlike PKB, the kinase activity of PDK1 is not influenced by binding to PtdIns(3,4,5)P3 in vitro, nor is it dependent on PI 3-kinase activity in the cell: Surprisingly, PDK1 is a constitutively active kinase that is neither stimulated by insulin nor inhibited by chemical inhibitors of PI 3-kinase. The dependence of PKB phosphorylation at Thr308 (and hence its activation) on PI 3-kinase activity lies at least in part in the binding of PtdIns(3,4,5)P3 to the PH domain of PKB, not to that of PDK1. PDK1 will only phosphorylate PtdIns(3,4,5)P3-bound PKB, suggesting that the PH domain of PKB normally restricts access of PDK1 to Thr308 but is displaced upon binding PtdIns(3,4,5)P3, allowing phosphorylation to occur. PI 3-kinase is necessary for other steps in the process—translocation of PKB to the plasma membrane (8) and the phosphorylation of Ser473 by PDK2 (9), which remains uncloned.

How do these reactions fit together to regulate PKB? A likely model is as follows: PtdIns(3,4,5)P3 binds to the PH domain of PKB, forcing its translocation to the plasma membrane and exposing Thr308 for phosphorylation by PDK1, which we now know is constitutively active. Serine-473 of PKB is then phosphorylated by PDK2, which is probably regulated by PtdIns(3,4,5)P3 and likely located at the plasma membrane, at least in its activated form. PDK1 itself appears to be localized to a large extent in the cytosol, but some of the enzyme undoubtedly will be at the plasma membrane as a result of its binding to PtdIns(3,4,5)P3. Concentration of all three of the kinases—PKB, PDK1, and PDK2—at the plasma membrane would facilitate the interactions of the upstream kinases with PKB. After its phosphorylation at both sites, PKB can detach from the membrane and phosphorylate its targets within the cell. PKB influences metabolism through phosphorylation of glycogen synthase kinase-3 and phosphofructokinase, as well as transmitting a potent survival signal. In part, this is by phosphorylation and inactivation of Bad, a pro-apoptotic Bcl-2 family member (10, 11), although other targets are likely.

In contrast, p70s6k participates in the translational control of mRNA transcripts containing 5′ polypyrimidine tracts. Regulation of p70s6k is also through PI 3-kinase, but it has appeared to be considerably more complex than that of PKB, involving phosphorylation of many more sites (12). However, the two kinases are distantly related, and p70s6k is phosphorylated at Thr229 and Thr389, sites that are analogous to Thr308 and Ser473 of PKB, the targets of PDK1 and PDK2. Pullen et al. (1) and Alessi et al. (4), therefore, tested whether PDK1 could phosphorylate p70s6k at Thr229 and found that indeed it could. Furthermore, a dominant negative form of PDK1 blocked activation of p70s6k in cells. This work represents the culmination of a huge effort that has led to the following model of p70s6k regulation: Initially proline-directed kinases such as mitogen-activated protein kinases phosphorylate a number of neighboring sites in the carboxyl-terminal autoinhibitory domain. This relieves a conformation inhibition that may result from interaction of the amino- and carboxyl-terminal portions of the protein. Thr389 is then phosphorylated by an unidentified PtdIns(3,4,5)P3-dependent kinase. Both of these steps are required to induce a conformational change that transforms Thr229 into a good substrate site for the constitutively active PDK1. As is the case for PKB and Thr308 and Ser473, phosphorylation of both sites causes a strongly synergistic activation of the kinase activity of p70s6k. As yet the identity of the kinase for Thr389 of p70s6k is unknown, but a sequence similarity with Ser473 of PKB suggests that PDK2 must be a good candidate. Phosphorylation of this site is PI 3-kinase dependent and reversed by a phosphatase indirectly activated by rapamycin.

The similarities in the regulation of these two distantly related kinases are quite striking. When inactive, both are in a conformation where the constitutively active PDK1 cannot phosphorylate its target site. In the case of PKB, a conformational change is elicited by binding of PtdIns(3,4,5)P3 to its PH domain, whereas for p70s6k this requires phosphorylation of the autoinhibitory domain by proline-directed kinases and phosphorylation of Thr389 by a PtdIns(3,4,5)P3-dependent kinase. After these changes, PDK1 can phosphorylate its target site. For PKB there may not be an obligatory order for the phosphorylation of the PDK1 and PDK2 sites. These findings neatly show how regulatory mechanisms have been conserved between distantly related members of the same family of kinases, and yet have been adapted to fit the special regulatory needs of each kinase. Because many more distantly related kinases have sites related to Thr308 of PKB, a family of PDK1-related enzymes could exist that control the activity of a great many kinases that can only be phosphorylated after a priming conformation changes.


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