Signal Transduction

Edited by: Steve Bunk H. Dudek, S.R. Datta, T.F. Franke, M.J. Birnbaum, R. Yao, G.M. Cooper, R.A. Segal, D.R. Kaplan, M.E. Greenberg, "Regulation of neuronal survival by the serine-threonine protein kinase Akt," Science, 275:661-5, Jan. 31, 1997. (Cited in more than 245 papers since publication) Alex Toker Comments by Henryk Dudek, senior scientist, neurosciences group, Ontogeny Inc., Cambridge, Mass., and Michael E. Greenberg, professor of neurology, Children's Hospital, Harvard Medical School T.F. Franke, D.R. Kaplan, L.C. Cantley, A. Toker, "Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate," Science, 275:665-8, Jan. 31, 1997. (Cited in more than 240 papers since publication) Comments by Thomas F. Franke, assistant professor of pharmacology, Columbia University, and Alex Toker, staff scientist, signal transduction group, Boston Biomedical Research Institute Among the most important realizations emerging from signal transduction research this decade has been the key role in cellular processes played by protein kinases. These enzymes catalyze phosphorylation, the addition of phosphate groups to proteins. In the complex cascade of communication links within one particular signaling pathway, such phosphorylation by a kinase called Akt is critical, as these two simultaneously published papers showed. Akt is a protein kinase encoded by the c-akt proto-oncogene, which is involved in normal cell growth but can also cause tumors when overexpressed. The first of the above two papers showed that Akt has a key role in sustaining the life of neuronal cells in the central nervous system. It demonstrated that insulin-like growth factor 1 (IGF-1) can activate an upstream kinase that already was known to be important to survival of various cell types. This enzyme, called phosphoinositide 3-kinase (PI3-K), triggers the activation of Akt. Further experiments established Akt as a critical mediator of neuronal survival. The second paper demonstrated that lipids, or fat-soluble products, of PI3-K bind directly to Akt in a domain called the pleckstrin homology (PH). These lipids act as "second messengers" by escorting Akt from the cytosol surrounding the nucleus to the cell membrane. There, they anchor Akt to the membrane by means of their binding at the PH domain. Akt then becomes activated by phosphorylation, and subsequently is released from the membrane, to itself phosphorylate other targets. In establishing a relationship between PI3-K, Akt, and cell survival, these two papers sparked a flurry of research that rapidly has implicated the PI3-K/Akt pathway in numerous biological processes, including both the programmed death of cells, or apoptosis, and its prevention. "I think Akt was a little bit of an orphan kinase," notes Henryk Dudek, who was lead author of the first paper as a postdoc in the lab of senior author Michael E. Greenberg. "There was a kinase of unknown function and a survival pathway with an unknown mediator. We put the two together." If putting them together was like connecting the knee bone to the foot bone, there were plenty of connections in between that were yet to be made. One point that remains in some contention is whether Akt is directly activated by binding to the lipid products of PI3-K. It is now known that the major mechanism for potent activation of Akt is its phosphorylation by PDK-1, an upstream kinase.1 "But the most critical thing about the action of Akt is its interaction with lipid," says Alex Toker, one of the authors of the second paper as a postdoc in Lewis C. Cantley's lab at Harvard Medical School. "For efficient Akt action, you must have lipid binding to initiate it." He adds that whether or not the lipids activate Akt in vivo, the paper's key point is that the lipids bind to Akt with high affinity and specificity, allowing for its subsequent phosphorylation. "I think that's why this paper has been cited so much: It gives a molecular explanation for Akt's activation." A molecular model is what was lacking in a 1995 Cell paper that preceded the later two studies. It not only identified Akt as a novel target of PI3-K but suggested the significance of both the Akt PH domain and lipid binding.2 Thomas F. Franke was lead author of the Cell paper as a postdoc in the lab of David R. Kaplan in Montreal Neurological Institute at McGill University, and later, was lead author of the first of the above two papers. "We've started something that now is getting into the clinical fields," he says, "where it becomes an interesting target for pharmacological development." In recent years, investigations have been intense into what Akt targets after its own activation. About a dozen such targets have been identified thus far, two of them by Greenberg's group. "Why there are so many targets of Akt is a question," he muses. "Akt certainly is one of the key enzymes, but evidence could emerge in a year or so that these targets are convergence points for other kinases as well." The first Akt target his lab found was a member of the BCL2 family of proteins, which includes both inhibitors and promoters of programmed cell death. The specific protein, called BAD (BCL2 antagonist of cell death), is a crucial component of the cell's apoptotic machinery. Akt phosphorylates BAD, blocking its ability to induce neuronal death.3 Another apoptotic target was found by a group under the leadership of John C. Reed, director of the apoptosis and cell death program at the Burnham Institute in La Jolla, Calif. It is caspase-9, an intracellular protease that is an effector of cell death. This paper was the first to show that caspases could be directly regulated by protein phosphorylation.4 Recently, Greenberg and colleagues pinpointed yet another Akt substrate. A member of the Forkhead transcription family, this target (called FKHRL1) differs from the other two, which are inhibited by Akt in a transcription-independent manner. Phosphorylation of FKHRL1 by Akt leads to retention of the transcription factor in the cytoplasm, where it is incapable of inducing apoptosis.5 Toker points out that Akt is ubiquitous, playing important roles not only in cell death or survival, but in insulin metabolism, among other functions. Indeed, the first Akt target ever identified, in 1995, was a kinase involved in control of glycogen and protein synthesis by insulin.6 "PI3-K also is implicated in many more biological processes which could be mediated by Akt," Toker adds. Current work of the lab he heads includes examining the role of the pathway in metastases of breast and colon cancer cells. Dudek observes, "In some situations, you might want to activate the pathway to promote neuronal survival and inhibit neurodegeneration. But in other situations, you might want to inhibit the pathway to prevent cancers." Recently, numerous papers have noted that mutations in a widely expressed tumor suppressor gene called PTEN promote high levels of active Akt in cancer cells. PTEN codes for a phosphatase, an enzyme that cleaves an inorganic phosphate from a compound. The normal PTEN phosphatase, which has an as-yet poorly understood role in controlling many types of tumors, apparently inactivates P13-K lipid products, thus inhibiting Akt. Last month, Reed's team established that another phosphatase, called calcineurin, can induce apoptosis by regulating the phosphorylation of BAD.7 Their experiments were done in part with hippocampal neurons, which are often affected in neurodegenerative disease. They found that the drug cyclosporine, commonly used to prevent organ rejection after transplantation, inhibited calcineurin and thus blocked BAD-induced apoptosis in hippocampal neurons. "Activating Akt to make it more potent is a nice concept, but there's no real pharmacologically valuable activator existent at this time," comments Franke, who collaborated with Reed's group. "We are trying to develop compounds now that are specifically effective for blocking Akt inhibitors." D.R. Alessi et al., "Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B *, " Current Biology, 7:261-9, 1997. T.F. Franke et al., "The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase," Cell, 81:727-36, 1995. S.R. Datta et al., "Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery," Cell, 91:231-41, 1997. M.H. Cardone et al., "Regulation of cell death protease caspase-9 by phosphorylation," Science, 282:1318-21, Nov. 13, 1998. A. Brunet et al., "Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor," Cell, 96:857-68, March 19, 1999. D.A. Cross et al., "Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B," Nature, 378:785-9, 1995. H.G. Wang et al., "Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD," Science, 284:339-43, April 9, 1999.

Signal Transduction

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