LONDON — The enzyme protein kinase B (PKB) is an object of great interest. All kinases add phosphates to other molecules, regulating enzymes or creating other molecules that act as intracellular messengers.
PKB plays a key role in many cellular events, from the inhibition of apoptosis and the control of cellular glucose levels to the regulation of the cell cycle. Being able to activate PKB could influence many biologically and medically important events, including the treatment of cancer — although no one is quite sure yet what kinds of drugs would have to be designed in order to modify its function.
Targeting therapies at PKB is thus a long way off. But little by little, the basic science that needs to be in place in order to work out how to manipulate this enzyme therapeutically is falling into position. The latest information, in two papers that have just appeared in Science, dated Jan. 30, 1998, is helping scientists to formulate a hypothesis about how PKB becomes activated.
One recently revealed clue to this process, reported in Cell last year (Vol 81:727-736), is that PKB can be activated by receptors found on the cell surface that also are capable of activating the phosphoinositide 3 kinases (PI 3 kinases). If the PI 3 kinases were inactivated, the activation of PKB also was inhibited.
Latest Research Builds On Previous Studies
Following this finding, researchers began to study how the lipid products of the PI 3 kinases — molecules called phosphotidylinositol 3,4,5-trisphosphate (Ptdins(3,4,5)P3) and phosphotidylinositol 3,4 bisphosphate (Ptdins(3,4)P2) — were able to activate an enzyme like PKB.
One theory was that Ptdins(3,4,5)P3 and Ptdins(3,4)P2 could activate PKB by binding to a region of this enzyme called the plekstrin-homology domain.
Earlier studies indeed had shown that phospholipids such as these could bind to the plekstrin-homology domain.
But Dario Alessi, of Dundee University, in Scotland, showed last year that the activation of PKB involves two critical phosphorylations, which are both inhibited if PI 3 kinase is inhibited (EMBO Journal 1996; Vol. 15:6541). These phosphorylations were on the threonine308 and the serine473 residues of PKB.
David Stokoe, of the University of California at San Francisco, and Len Stephens, of the Babraham Institute, in Cambridge, U.K., then went on to identify an enzyme which could phosphorylate PKB on threonine308, leading to the activation of PKB. Stokoe and his colleagues, who reported this work in Science (Vol. 227:567-570), called this enzyme PKB kinase, although it has now been named PDK1.
They also showed that, in the test tube, Ptdins(3,4,5)P3 and Ptdins(3,4)P2 could stimulate phosphorylation of PKB and hence its activation. They therefore proposed that, in vivo, cell surface receptors were activating PI 3 kinase, leading to the production of Ptdins(3,4,5)P3 and Ptdins(3,4)P2, which in turn activated PDK1 to phosphorylate and thus activate PKB.
All this sets the scene for the two Jan. 30 Science papers. Stephens, head of the Inositide Laboratory at the Babraham Institute, and colleagues, in a paper titled "Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B," describe how they have cloned the gene for PDK1 and expressed it in cultured cells. The product of this gene is a protein kinase that can phosphorylate PKB at threonine308, and this phosphorylation is activated by Ptdins(3,4,5)P3.
Stephens told BioWorld International, "This latest piece of information allows us to put together a hypothesis about how PKB becomes activated. We suggest that when PI 3 kinase becomes activated, it makes the phospholipids Ptdins(3,4,5)P3 and Ptdins(3,4)P2, which — being phospholipids — are synthesized on the inside of the plasma membrane and stay there. There, they act as specific signals to cause the recruitment of both PDK1 and PKB from the cytosol to the plasma membrane. These molecules bind to the phospholipids at their plekstrin-homology domains, and they are thus co-localized at the membrane.
"So Ptdins(3,4,5)P3 operates in a complex fashion, causing the translocation of two different enzymes, one of which acts as a substrate for the other, into the same cell-surface position."
The second Science paper, by N. Pullen and colleagues, of the Friedrich Miescher Institute, in Basel, Switzerland, is titled "Phosphorylation and activation of p70s6K by PDK1." Their work shows that the protein kinase p70 ribosomal protein S6 kinase (p70s6K) also is regulated by PDK1, which phosphorylates it at threonine229.
Commenting on the two papers in the same issue of Science, Julian Downward, of the Signal Transduction Laboratory at the Imperial Cancer Research Fund, in London, said: "The similarities in the regulation of these two distantly related kinases are quite striking. These findings neatly show how regulatory mechanisms have been conserved between distantly related members of the same family of kinases."
Stephens and his colleagues, who are collaborating with Onyx Pharmaceuticals Inc., of Richmond, Calif., have applied for a patent protecting the use of PKB kinase as a potential therapeutic or diagnostic agent.
The next problem they plan to address is how the activation of PKB leads to an increased phosphorylation of its substrates — and, particularly, where this event occurs in the cell.